Methods of expanding and assessing b cells and using expanded b cells to treat disease

ABSTRACT

Provided herein are methods of expanding B cells, and in particularly B10 cells capable of producing IL-10, ex vivo. The methods include incubation of harvested B cells in the presence of IL-21. Compositions comprising the ex vivo expanded B cells and methods of using the expanded B cell-containing compositions to treat diseases or conditions are also provided. Methods of assessing B10 cell function in a subject are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 16/000,604, filed Jun. 5, 2018, and issued as U.S. Pat. No.10,611,999 on Apr. 7, 2020, which claims the benefit of priority of U.S.patent application Ser. No. 13/795,889, filed Mar. 12, 2013, and issuedas U.S. Pat. No. 10,017,739 on Jul. 10, 2018, which claims the benefitof priority of U.S. Provisional Patent Application No. 61/697,663, filedSep. 6, 2012 and U.S. Provisional Patent Application No. 61/707,256,filed Sep. 28, 2012, all of which are incorporated herein by referencein their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support awarded bythe National Institutes of Health grant number A1057157 and U19 A156363.The United States may have certain rights in this invention.

INTRODUCTION

It is well-known that B cells regulate immune responses by producingantigen-specific antibody. However, specific B cell subsets can alsonegatively regulate immune responses, validating the existence ofregulatory B cells. Human and mouse regulatory B cells (B10 cells) withthe ability to express the inhibitory cytokine interleukin-10 (IL-10)have been identified. Although rare, B10 cells are potent negativeregulators of antigen-specific inflammation and T cell-dependentautoimmune disease in mice. B10 cell IL-10 production regulatesantigen-specific immune responses in vivo without inducing systemicimmunosuppression. B10 cells may thereby be useful in regulating orcontrolling inflammation or autoimmune diseases.

SUMMARY

Provided herein are methods of expanding B cells ex vivo, compositionscomprising expanded B cells and methods of using the expanded B cellcompositions for assessing or screening for a disease state or conditionand for treating diseases as described herein. Methods of expanding B10cells capable of producing IL-10 ex vivo by culturing or incubating Bcells harvested and isolated from a subject with IL-21 are providedherein. The resultant expanded polyclonal B cells can be collected orisolated from the culture and may be further harvested to select for B10cells.

The methods of expanding B cells ex vivo include culturing B cellsharvested from a subject on feeder cells expressing a CD40 agonist and aB cell survival promoter such as BAFF in the presence of IL-4 and thenculturing the resultant cells on feeder cells expressing a CD40 agonistand a B cell survival promoter such as BAFF in the presence of IL-21prior to collecting or isolating the expanded polyclonal B cells. Theexpanded polyclonal B cells may also be further selected. For example,the expanded polyclonal B cells may be further selected to isolate B10cells.

Compositions comprising the expanded polyclonal B cells and B10 cellsproduced by the methods described herein are also provided. Thecomposition comprising the expanded polyclonal B cells produced by themethods described herein may be further selected to produce acomposition comprising B10 cells. The compositions of expandedpolyclonal B cells and/or B10 cells may be used in a variety of methodsto treat various diseases or conditions. Pharmaceutical compositionsincluding the expanded polyclonal B cell and B10 cell compositionsdescribed herein are also provided.

Methods of treating a subject having an autoimmune disease, an allergicdisorder, an inflammatory disorder or immunodeficiency are provided. Themethods include administering a therapeutically effective amount of thecompositions comprising expanded polyclonal B cells or B10 cellsdescribed herein to a subject in need of treatment for an autoimmunedisease, an allergic disorder, an inflammatory disorder or animmunodeficiency.

Methods of treating a subject to prevent or treat organ, tissue or celltransplant rejection or associated graft versus host disease are alsoprovided. The methods include administering a therapeutically effectiveamount of the compositions including the B cells and/or B10 cellsdescribed herein to a subject in need of treatment for transplantrejection or graft versus host disease.

Methods of treating a subject receiving recombinant, therapeutic orxenogeneic protein(s) are also provided. The methods includeadministering a therapeutically effective amount of the compositionsincluding the B cells and/or B10 cells disclosed herein to a subject inneed of treatment for a genetic, transplantation, allergy, inflammation,or autoimmune disorder.

Methods of assessing B10 cell function in a subject are also provided.In these methods, the B cells are harvested from a sample from thesubject and cultured in the presence of IL-21 for at least 24 hours. TheB cells are then tested to determine whether the cells are capable ofproducing IL-10 and/or the amount of IL-10 produced or the percentage ofcells capable of producing IL-10 in the culture as compared to a controlhaving normal B cell function is determined. B cells expressing IL-10are B10 cells. The B cells may be cultured on feeder cells expressing aCD40 agonist and a B cell survival promoter such as BAFF in the presenceof IL-4 and subsequently in the presence of IL-21 prior to assessment ofthe ability of the cells to produce IL-10.

The methods of assessing B10 cell function provided herein may be usedto diagnose an autoimmune or inflammatory disease or may be used toassess the stage of a disease or condition in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of data which demonstrate that IL-21 induces regulatoryB10 cell function. (A) FACS analysis dot plots and graphs of the datashowing that IL-21 induces B10 cell IL-10 production and secretion.Spleen CD19⁺ B cells purified from wild type mice were cultured inmedium alone or with the indicated recombinant cytokines or LPS. Tovisualize IL-10-competent B cells, LPS, PMA, ionomycin and monensin(L+PIM) were added to the cultures 5 h before the cells were isolated,stained with CD19 mAb, permeabilized, stained for cytoplasmic IL-10expression and analyzed by flow cytometry. Representative histogramsshow IL-10⁺ cell frequencies within the indicated gates, with backgroundstaining shown for cells cultured with monensin (Mon.) alone. Bar graphsindicate mean (±SEM) IL-10⁺ B cell frequencies or culture supernatantfluid IL-10 concentrations at 48 or 72 h from three independentexperiments using individual mice. (B) Dot plots showing that IL-21induces CD1d^(hi)CD5⁺ B cell IL-10 production. Purified spleenCD1d^(hi)CD5⁺ or CD1d^(lo)CD5⁻ B cells from wild type mice were culturedwith media alone or containing IL-21 for 48 h before IL-10⁺ B cellfrequencies were assessed as in (A). (C) Graphs showing that B10 cellsexpress IL-21R. CD19⁺ splenocytes purified from wild type mice werecultured with L+PIM for 5 h before cell surface CD19 and IL-21R, andcytoplasmic IL-10 staining to identify IL-10-competent B10 cells (dotplot, left panel). Representative IL-21R expression by IL-10⁺ and IL-10⁻B cells from wild type mice is shown in comparison with control B cellsfrom IL-21R^(−/−) mice (gray histograms). These results arerepresentative of three independent experiments using individual mice.(D) Graphs showing that IL-21R expression is required for B10 cellexpansion in vivo following MOG immunization. B10 cell numbers wereassessed in wild type, IL-21R^(−/−) or CD19^(−/−) mice 7 days afterreceiving PBS or MOG₃₅₋₅₅ immunization. Representative flow cytometryhistograms are shown. Bar graphs indicate mean (±SEM) B10 cellfrequencies (>3 mice per group). Significant differences between samplemeans are indicated in (A) and (D); *, p<0.05; **, p<0.01.

FIG. 2 is a set of data which demonstrate that B10 cells require IL-10,IL-21R, CD40, and MHC-II expression to regulate EAE severity. (A)Experimental protocol and resulting disease severity in various miceafter administration of MOG₃₅₋₅₅. One day before CD19^(−/−) or wild type(WT) mice were immunized with MOG₃₅₋₅₅ on day 0, the CD19^(−/−) micereceived saline (PBS) or purified spleen CD1d^(hi)CD5⁺ or CD1d^(lo)CD5⁻B cells from either wild type, IL-10^(−/−), IL-21R^(−/−), CD40^(−/−), orMHC-II^(−/−) mice. The mice were scored daily thereafter for diseaseseverity.

The top two graphs show data from the same experiment, but wereseparated since the curves superimposed and thus were difficult tovisualize on a single graph. (B) Graphs showing that B10 cells requireMHC-II expression to regulate EAE severity in wild type mice treatedwith CD20 or control mAb 7 days before MOG₃₅₋₅₅ immunization on day 0.The mice also received PBS or purified CD1d^(hi)CD5⁺ B cells from eitherCD20^(−/−) or MHC-II^(−/−) CD20^(−/−) mice 1 day before MOG₃₅₋₅₅immunization. The two graphs are from the same experiment, but wereseparated to facilitate visualization of the overlapping curves. (C)Graph showing that activated MHC-II^(−/−) B10 cells are not able toreduce disease severity in wild type mice. Purified CD1d^(hi)CD5⁺ Bcells from wild type or MHC-II^(−/−) mice were cultured with agonisticCD40 mAb for 48 h to induce B10pro cell maturation, with LPS addedduring the final 5 h of culture. Wild type mice were given either PBS orCD1d^(hi)CD5⁺ B cells 1 day before MOG₃₅₋₅₅ immunization on day 0. In(A)-(C) values represent mean (±SEM) symptom scores from >3 mice in eachgroup, with similar results obtained in three independent experiments.Significant differences between sample means are indicated; *, p<0.05.

FIG. 3 is a set of data showing B10 cell expansion and regulation of Tcell-mediated autoimmunity. (A) Dot plots and graphs showing that B10cells require IL-10, IL-21R, CD40 and MHC-II expression to regulateantigen-specific T cell proliferation in vivo. CD19^(−/−) recipient micewere given PBS as a control, or purified CD1d^(hi)CD5⁺ or CD1d^(lo)CD5⁻B cells from naïve wild type (WT), IL-10^(−/−), IL-21R^(−/−), CD40^(−/−)or MHC-II^(−/−) mice, or wild type mice with EAE (day 28) 1 day beforeMOG₃₅₋₅₅ immunization on day 0. Four days after immunization, dye(CFSE)-labeled TCR^(MOG) CD4⁺ Thy1.1⁺ T cells were transferred intoCD19^(−/−) recipient mice. Five days later, peripheral lymph node CD4⁺Thy1.1⁺ T cells were analyzed for proliferation, with representativeflow cytometry analysis of CFSE dilution shown. Bar graphs indicate mean(±SEM) numbers of divided TCR^(MOG) T cells. (B) Dot plots and graphsshowing that B10 cells require IL-10, IL-21R, CD40 and MHC-II expressionfor their regulation of antigen-specific T cell cytokine production.Purified CD1d^(hi)CD5⁺ B cells from the indicated mice were transferredinto CD19^(−/−) recipient mice 1 day before MOG₃₅₋₅₅ immunization on day0, with TCR^(MOG) Thy1.1⁺ CD4⁺ T cells transferred on day 4. Fourteendays later, lymph node Thy1.1⁺ CD4⁺ T cells were analyzed for IL-17 andIFN-γ production by intracellular cytokine staining, with representativeflow cytometry results shown. Bar graphs indicate mean (±SEM)frequencies of cytokine-expressing cells, with three mice in each group.In (A) and (B), significant differences between sample means areindicated: *, p<0.05; **, p<0.01. (C) A model for autoantigen(Ag)-specific B10 cell function. B cells capture autoantigens thattrigger appropriate BCR signals (step 1) and promote IL-10-competentB10pro cell development. During immune responses (step 2), B10pro cellspresent peptides to antigen-specific T cells through cognateinteractions that induce T cell activation and CD40/CD154 interactions.Activated T cells may produce IL-21 locally, which binds to proximal B10cell IL-21R (step 3). IL-21R signals induce B10 cell IL-10 productionand effector function (B10eff, step 4), which may negatively regulateantigen-specific T cell function (step 5).

FIG. 4 is a set of data showing that IL-21 drives ex vivo regulatory B10cell expansion. (A) Dot plots showing B10 cell development in vitro.Purified spleen B cells were cultured on a monolayer of NIH-3T3 cellsexpressing CD154/BLyS in the presence of exogenous IL-4 for 4 days, thencultured on fresh NIH-3T3-CD154/BLyS cells with exogenous IL-21 for 3 or5 days as indicated. The cells were then isolated, cultured withmonensin for 5 h and stained for cytoplasmic IL-10 expression.Representative IL-10⁺ B cell frequencies within the indicated gates areshown. Similar results were obtained in ≥10 experiments. (B) Asuperimposed bar and line graph showing that IL-21 drives B10 cellexpansion in vitro. B cells were cultured as in (A) with cells harvestedeach day of culture. Bar values represent mean (±SEM) B cell and B10cell numbers, or B10 cell frequencies (solid line) from threeindependent experiments. (C) Dot plots showing IL-21-induced B10 cellsto express CD5. Wild type B cells were cultured for 9 days as in (A) andstained for CD5 and CD19 expression. CD5⁺ and CD5⁻ B cells were thenpurified by cell sorting and cultured with monensin for 5 h beforecytoplasmic IL-10 staining. Results shown are representative of threeindependent experiments (D) Graphs showing that IL-21-induced B10effector cells inhibit EAE initiation and progression. IL-21-induced B10cells (CD5⁺ CD19⁺) or non-B10 cells (CD5⁻ CD19⁺) were isolated as in (C)and adoptively transferred into wild type mice on days −1, 7, 14 or 21(arrows) before/after MOG immunization and EAE induction as in FIG. 2.(E) Bar graph showing that B10 cell expansion in vitro requires IL-21Rand CD40 expression, and in vivo BCR signaling. Purified spleen B cellsisolated from wild type, IL-21R^(−/−), CD40^(−/−), MHC-II^(−/−),CD19^(−/−), or MD4 mice were cultured as in (A), with mean (±SEM) cellnumbers quantified after culture. Values represent means (±SEM) of threeindependent experiments. IL-10⁺ B cell frequencies in the cultures areshown in parentheses. (F) Graphs showing that IL-21-induced B10 cellsrequire IL-10 and MHC-II expression to inhibit EAE. B cells fromIL-10^(−/−) or MHC-II^(−/−) mice were cultured as in (A), separated intoCD5⁺ or CD5⁻ cells as in (C) and adoptively transferred into wild typemice before MOG₃₅₋₅₅ immunization as in (D). In (D) and (F), valuesrepresent mean (±SEM) symptom scores from ≥3 mice in each group, withsimilar results obtained in three independent experiments. In (B), (D)and (E), significant differences between sample means are indicated: *,p<0.05; **, p<0.01.

FIG. 5 presents a set of data showing that IL-21 induces regulatory B10cell function. (A) Bar graphs showing that IL-21 induces B10 cell IL-10production and secretion. Spleen CD19⁺ B cells purified from wild typemice were cultured with medium alone or with the indicated cytokines for48 or 72 h. To visualize IL-10-competent B cells, monensin was added tothe cultures 5 h before the cells were isolated, stained with CD19 mAb,permeabilized, stained for cytoplasmic IL-10 expression and analyzed byflow cytometry. Bar graphs indicate mean (±SEM) IL-10⁺ B cellfrequencies or numbers at 48 and 72 h from individual mice in threeindependent experiments. Significant differences between media versuscytokine sample means are indicated: *, p<0.05; **, p<0.01. (B-D) Dotplots and bar graphs showing IL-21R, CD40 and MHC-II expression are notrequired for B10 or B10pro cell development, respectively. Purifiedspleen B cells from wild type and IL-21R^(−/−) (B) CD40^(−/−) (C) orMHC-II^(−/−) (D) mice were cultured with monensin alone or L+PIM for 5 hto quantify B10 cell frequencies. Alternatively, B10+B10pro cellfrequencies were determined after culturing the cells ex vivo withagonistic CD40 mAb for 48 h, with L+PIM added during the final 5 h ofculture. Representative histograms and bar graphs indicate mean (±SEM;≥3 mice per group) percentages and numbers of B cells that expressedIL-10 in one of two experiments with equivalent results.

FIG. 6 is a set of dot blots and bar graphs showing that T follicularhelper cells are present in CD19^(−/−) mice. Representative flowcytometry analysis of CXCR5^(hi)PD1⁺ cells among spleen CD4⁺ T cellsfrom wild type and CD19^(−/−) mice. Bar values represent mean (±SEM)CXCR5^(hi)PD1⁺ cell frequencies among CD4⁺ T cells from three mice.Significant differences between sample means are indicated: *, p<0.05.

FIG. 7 is a graph showing the total number of B cells and B10 cellsafter the indicated time in culture and the amount of IL-10 produced bythe culture. Purified human blood B cells were cultured onNIH-3T3-mCD154/hBLyS cell monolayers with exogenous human IL-4 (2 ng/ml)for 7 days. Additional media containing IL-4 (2 ng/ml) was added to thecultures on days 2 and 4. The B cells were then isolated and cultured onfresh NIH-3T3-CD154/BLyS cells with exogenous human IL-21 (10 ng/ml) for5 days as indicated. The cells were then isolated, cultured with CpG+PIBfor 5 h and stained for cell surface CD19 and cytoplasmic IL-10expression. Bar values represent mean (±SEM) CD19⁺ B cell and B10 cellnumbers, or B10 cell frequencies (solid line) from two independentexperiments.

DETAILED DESCRIPTION

The B10 cell subset of regulatory B cells has been functionally definedin humans and mice by their ability to express IL-10. B cells that arecompetent to express IL-10 following 5 h of ex vivo phorbol ester andionomycin stimulation are functionally defined as B10 cells todistinguish them from other regulatory B cells that modulate immuneresponses through other mechanisms. B10 cells are found at lowfrequencies (1-5%) in naïve mice but expand with autoimmunity. SpleenB10 cells are predominantly found within the minor CD1d^(hi)CD5⁺ B cellsubpopulation along with rare B10 progenitor (B10pro) cells that areinduced to become IL-10-competent during in vitro culture with agonisticCD40 monoclonal antibody (mAb). The capacity of human and mouse B10cells to produce IL-10 is central to their ability to negativelyregulate inflammation and autoimmune disease, as well as adaptive andinnate immune responses, but the physiologic signals that control IL-10production in vivo are unknown.

B10 cell immunoregulation is antigen-specific, and B cell antigenreceptor (BCR) specificity dramatically influences B10 cell development.Receptors or pathways that positively or negatively regulate BCRsignaling can also modulate B10 cell numbers in vivo. For example,CD19-deficient (CD19^(−/−)) mice are essentially devoid of regulatoryB10 cells, which leads to exacerbated inflammation and disease symptomsduring contact hypersensitivity and in the experimental autoimmuneencephalomyelitis (EAE) model of multiple sclerosis. IL-10 itself is notrequired for B10 cell development since B cells with the capacity toexpress IL-10 reporter genes develop normally in IL-10^(−/−) mice. B10cell numbers are also normal in T cell-deficient nude mice and in micedeficient in expression of major histocompatibility complex class II(MHC-II) or CD40 molecules that are important for cognate B cell-T cellinteractions. Consequently, appropriate BCR signals are thought toselect a subset of B cells to become IL-10-competent B10 cells. Innatepathogen-induced signals also influence regulatory B10 cell developmentin vivo. Little is otherwise known about how B10 cell IL-10 productionis regulated in vivo, and it remains unclear how such rare B cells exertsuch potent in vivo effects and selectively inhibit antigen-specific Tcell function during inflammation and autoimmunity.

Using a mouse model for multiple sclerosis, we show here that B10 cellmaturation into functional IL-10-secreting effector cells that inhibitin vivo autoimmune disease requires IL-21 and CD40-dependent cognateinteractions with T cells. Moreover, the ex vivo provision of CD40 andIL-21 receptor signals can drive B10 cell development and expansion byup to four-million-fold and generate B10 effector cells producing IL-10that dramatically inhibit disease symptoms when transferred into micewith established autoimmune disease. Thereby, the ex vivo expansion andreinfusion of autologous B10 cells may provide a novel and effective invivo treatment for autoimmune diseases and other conditions that areresistant to current therapies.

In addition, we also show that human B cells and B10 cells can beexpanded ex vivo. The B cells were harvested from normal human blood andexpanded ex vivo using the same methods as were used for expansion ofmouse B cells. As described in the Examples, B cell numbers wereincreased by 130 fold while B10 cells were increased by 5-6,000 fold.Thus, the examples demonstrate that the methods may be used to generateex vivo expanded B cells that may be useful for autologous treatment ofhuman diseases or conditions in which addition of responsive B cells orof B10 cells may be therapeutic.

Methods of Expanding B Cells Ex Vivo

Described herein are methods of expanding polyclonal B cells andspecifically B10 cells ex vivo. The methods include harvesting B cellsfrom a subject and incubating them with IL-21. In FIG. 1, the B10 cellswere expanded over two fold after 48 hours of ex vivo incubation withIL-21 at 100 ng/ml and over three fold after 72 hours incubation. Themethods may further include incubation with a CD40 ligand, and a B cellsurvival promoter such as BAFF (BLyS, used interchangeably herein) orfeeder cells expressing a CD40 ligand and/or BAFF to result in furtherexpansion of B cells and B10 cells. As shown in the Examples, the Bcells may be further expanded ex vivo by a first incubation with IL-4followed by a second incubation including IL-21. Either or both of thesesteps may include feeder cells and optionally a CD40 ligand and/or BAFF.Total B cells were expanded using the methods described herein, but B10cells were expanded at a higher frequency using these methods. Inaddition, the B10 cells were able to produce IL-10 without a need forfurther stimulation ex vivo with LPS or another stimulatory signal.Suitably, the B cells are human B cells.

In an alternative embodiment of the methods of expanding B cellsdescribed herein, the B cells are harvested from a subject and thenincubated on feeder cells expressing a CD40 agonist and a B cellsurvival promoter such as BAFF in the presence of IL-4. This incubationperiod may last from two to ten or more days and the amount of IL-4 maybe optimized. In the methods, 2 ng/ml IL-4 was used, but 0.5 ng/ml to100 ng/ml may be useful. The resultant cells were then incubated on thefeeder cells expressing CD40 agonist and a B cell survival promoter suchas BAFF in the presence of IL-21 for an additional two to eight or moredays before harvesting the expanded B cells. In the Examples, either 10ng/ml or 100 ng/ml of IL-21 was used, but between 5 ng/ml and 1000 ng/mlof IL-21 may be used. The actual amount of IL-4 and IL-21 used in themethods can be determined by those of skill in the art and will dependon the culture conditions, including whether the culture media and thecytokines were replenished over time, the length of the culture periodand other culture conditions. In the Examples, total polyclonal B cellsand B10 cells were expanded using this method. However, the cultures maystart with single B cells or isolated B cell subsets that are thenexpanded ex vivo into monoclonal, pauciclonal or polyclonal B cellpopulations. The B cells produce antibodies under the culture conditionsdescribed herein and thus the methods may be used to select for amonoclonal, pauciclonal or polyclonal population of antibody producing Bcells. Suitably, the B cells are mouse B cells, Suitably the B cells arehuman B cells.

The B cells used in the methods may be harvested from various areas ofthe subject, including but not limited to the blood, spleen, peritonealcavity, lymph nodes, bone marrow, site of autoimmune disease, site ofinflammation or tissue undergoing transplant rejection in the subject.The cells may be harvested from the subject by any means available tothose of skill in the art. The harvested population of cells shouldcontain B cells, but may be a mixed cellular population. The subject maybe any animal with B lymphocytes, suitably a mammal, suitably adomesticated animal such as a horse, cow, pig, cat, dog, or chicken, orsuitably a human. Alternatively, the cells may be derived from stemcells, including but not limited to B cell stem cells, bone marrow stemcells, embryonic stem cells and induced pluripotent stem cells, whichhave been appropriately differentiated in vitro to develop into B cellsor B cell progenitors prior to use in the methods described herein. Seee.g., Carpenter et al., 2011, Blood 117: 4008-4011.

The B cells may be isolated from the subject by removal of non-B cells,or selection for cell surface markers such as IgM, IgD, IgG, IgA, IgE,CD19, CD20, CD21, CD22, CD24, CD40, CD72, CD79a or CD79b, orcombinations of these cell surface molecules including CD1d, CD5, CD9,CD10, CD23, CD27, CD38, CD48, CD80, CD86, CD138 or CD148. The expanded Bcells may be harvested by selecting for these markers after ex vivoculturing in the method or specific B cell types, such as B10 cells, maybe selected using these markers before or after ex vivo expansion eitheralone or in combination. The B10 cells may be harvested by selecting forCD1d, CD5, CD24, CD27 or combinations thereof. In some embodiments, theB10 cells are capable of producing IL-10 after incubation with IL-21 andthus may be selected, isolated or harvested by selecting for IL-10production. In other embodiments, the B10 cells may need to be furtherstimulated to produce IL-10 with e.g., LPS or PMA and ionomycin. Methodsof stimulating B10 cells to produce IL-10 are known in the art andinclude stimulation with LPS or CpG oligonucleotides.

As used herein expansion of B cells includes stimulation ofproliferation of the cells as well as prevention of apoptosis or otherdeath of the cells. As used herein, “culturing” and “incubation” areused to indicate that the cells are maintained in cell culture medium at37° C. and 5% CO₂ for a period of time with the indicated additives(feeder cells, cytokines, agonists, other stimulatory molecules ormedia, which may include buffers, salts, sugars, serum or various otherconstituents). Suitably, the incubation or culturing periods used hereinis at least 48 hours, but may be for any amount of time up to eight ormore days. As shown in the Examples more than one culturing period maybe used. In the Examples, for mouse B cell expansion the culture withIL-4 was four days long and the culture with IL-21 was five days. Forhuman B cells, the expansion with IL-4 was a seven day culture periodfollowed by a five day culture with IL-21. Those of skill in the artwill appreciate that the culturing or incubation time may be varied toallow proper expansion, to adjust for different cell densities orfrequencies of individual subsets, and to allow an investigator toproperly time use of the cells. Thus the precise culture length may bedetermined empirically by one of skill in the art.

As used herein, isolating is used to indicate that a group of cells isseparated from incubation media, feeder cells or other non-B cells.Isolating is not meant to convey that the resulting isolated cells havea certain level of purity or homogeneity. The cells may be harvested,isolated or selected using any means available to those of skill in theart. For example, B cells may be harvested from adherent cells byselecting for non-adherent cells after an appropriate incubation. Cellsmay also be selected for expression of cell surface markers by FACSsorting or by the differential ability to bind antibody coated magneticbeads. Means of selecting cells in a mixed population are well known tothose skilled in the art.

Non-limiting examples of CD40 agonists include CD40 antibodies andfragments thereof, the CD40 ligand (CD154) and polypeptide fragmentsthereof, small molecules, synthetic drugs, peptides (including cyclicpeptides), polypeptides, proteins, nucleic acids, aptamers, synthetic ornatural inorganic molecules, mimetic agents, and synthetic or naturalorganic molecules. In a certain embodiment, the CD40 agonist is a CD40antibody. The CD40 antibodies can be of any form. Antibodies to CD40 areknown in the art (see, e.g., Buhtoiarov et al., 2005, J. Immunol.174:6013-22; Francisco et al., 2000, Cancer Res. 60:3225-31; Schwulst etal., 2006, 177:557-65, herein incorporated by reference in theirentireties). The CD40 agonists may be CD40 ligands and may be expressedon the surface of feeder cells or soluble.

The BAFF (BLyS) may be expressed by the feeder cells via methods knownto those of skill in the art. The BLyS may be expressed on the surfaceor may be soluble after cleaved from the cell surface. Alternatively theBAFF is replaced by a different factor(s) that promotes B cell survivalin culture including feeder cells, BAFF fragments, APRIL, CD22 ligand,CD22 monoclonal antibody, or fragments thereof.

The feeder cells used in the Examples were fibroblasts but other feedercells may be used in the methods. The feeder cells may be endothelialcells, epithelial cells, keratinocytes, melanocytes, or othermesenchymal or stromal cells. The incubation or culturing periods usedin the methods may be from two to ten or more days for each step in themethod. Suitably, the incubation time is between three and seven days,suitably it is between four and five days. As described in the examplesthe feeder cells are likely required to supply additional signals, otherthan the CD40 agonist and BAFF, to allow optimal B cell expansion in themethods. A preliminary analysis of other factors supplied by the feedercells to optimize B cell expansion is included in the Examples and Table1 below. In summary, in addition to a CD40 agonist and BAFF, the feedercells minimally express VCAM-1 and CD44 in addition to CD40 agonist andBAFF. Increased expression of CD24, interleukin-7 (IL-7), Mst1 and Tslpby the feeder cells correlated with the feeder cells being capable ofproducing increased numbers of B cells during ex vivo expansion.Similarly, downregulation of certain molecules in the feeder cellscorrelated with increased ability to support B cell expansion. Inparticular, downregulation of CD99, TGFBI, CXCR7, Dlk1, Jag1 and Notch1on the feeder cells correlated with the cells being better capable ofsupporting B cell expansion ex vivo. Thus, those of skill in the art maybe able to select for, or create via genetic engineering, feeder cellsbetter capable of supporting B cell expansion ex vivo. The informationmay also be used to generate a means of expanding B cells that does notrequire live feeder cells for optimal ex vivo B cell expansion.

The methods may allow from two fold to over 5×10⁶ fold expansion of Bcells or B10 cells in particular. The cells may be selected after theculture period to remove any non-B cells or to positively select for Bcells or for a particular B cell subset such as B10 cells. The B10 cellsmay represent 10%, 15%, 20%, 25%, 30%, 35%, 40% or more of the total Bcells in the culture after the expansion method is complete. Afterselecting the cells for cell surface expression of a B10 cell surfacemarker(s) more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ofthe cells are B10 or B10pro cells capable of producing IL-10.

Compositions Comprising the Expanded B Cells

Compositions including the expanded polyclonal B cells generated usingthe methods described herein are also provided. In one aspect, thecompositions include more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or substantially 100% B10 or B10pro cells. In otheraspects the compositions are selected to include antibody producing Bcells. The compositions may include more than 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 99% or substantially 100% antibodyproducing B cells.

The expanded B cell containing compositions may be used to makepharmaceutical compositions. Pharmaceutical compositions comprising theexpanded B cells described above and a pharmaceutically acceptablecarrier are provided. A pharmaceutically acceptable carrier is anycarrier suitable for in vivo administration of cells. Examples ofpharmaceutically acceptable carriers suitable for use in the compositioninclude, but are not limited to, buffered solutions, glucose solutions,oil-based or cellular culture based fluids. Additional components of thecompositions may suitably include, for example, excipients such asstabilizers, preservatives, diluents, emulsifiers and lubricants.Examples of pharmaceutically acceptable carriers or diluents includestabilizers such as carbohydrates (e.g., sorbitol, mannitol, starch,sucrose, glucose, dextran), proteins such as albumin or casein,protein-containing agents such as bovine serum or skimmed milk andbuffers (e.g., phosphate buffer).

The expanded B cell compositions may be co-administered with othertreatments, such as small molecule, polypeptide, antibody, aptamer orother therapeutics. Co-administration of the compositions describedherein with another therapeutic may be administered in any order, at thesame time or as part of a unitary composition. The two compositions maybe administered such that one composition is administered before theother with a difference in administration time of 1 hour, 2 hours, 4hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7days, 2 weeks, 4 weeks or more.

In another embodiment, the B cell or B10 cell population may bemonoclonal or pauciclonally expanded from isolated single cells orisolated B cell subsets. In one aspect, the compositions include morethan 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 99% or substantially 100% monoclonal Bcells or B10 cells. In other aspects the compositions are selected toinclude antibody producing B cells. The compositions may include morethan 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 99% or substantially 100% antibodyproducing B cells. In another aspect the compositions are selected toinclude monoclonal or pauciclonal antigen-specific B cells. Thecompositions may include more than 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% orsubstantially 100% B cells specific for or producing antibody against asingle protein or other antigenic entity. In another aspect, thecompositions may include more than 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99% or substantially 100% B cells capable ofexerting regulatory activities by expressing IL-113, IL-2, IL-4, IL-5,IL-6, IL-12, IL-13, IL-17, IFNγ, IL-23 or TNF-α.

In another embodiment, the B cell or B10 cell population can be maderesponsive to a certain antigen involved in a specific disease. The Bcells may produce therapeutic antibodies or other cytokines in responseto subsequent encounter with the antigen. The B10 cell population, whensensitized with a certain antigen, may produce therapeutic amounts ofIL-10 upon subsequent encounters with the antigen. Such antigen-specificB cell or B10 cell populations may be used in adoptive transfer methods,wherein a subject is or has previously been immunized with a certainantigen and the antigen-sensitized cells from said subject are isolated,expanded ex vivo by the methods described herein and transplanted to thesame or another subject. Alternatively, a B cell or B10 cell populationfrom a subject can be isolated and subsequently can be sensitized with adisease-specific antigen ex vivo or in vitro. The sensitized cellpopulation can then be transplanted into the original or another subjectby any method known in the art. In still another embodiment, theantigen-specific B cell or B10 cell population can be added to animplantable immune modulation device. According to this embodiment, theimplanted cell population will produce strategically localized IL-10,antibody or another cytokine production when encountering antigen in thehost, depending on the cells implanted. In a further aspect, the B cellor B10 cell population and a disease-specific antigen can both be placedin an implantable immune modulation device, and said device then can betransplanted into a recipient at a location where the therapeuticeffects of the cell population, i.e., IL-10 production, antibodyproduction or cytokine production, are needed, thus resulting in anamplified response to the disease in vivo.

In another aspect, a certain disease-specific antigen can beadministered in conjunction with a CD40 agonist or a TLR agonist. Thetherapeutic agent may be an antibody, in particular, a CD40 antibody orLPS or CpG oligodeoxynucleotides. In other aspects, the therapeuticagent is a small molecule, a polypeptide, DNA, or RNA that interactswith the B cell CD40 receptor or TLRs.

Any antigen from any disease, disorder, or condition may be used inaccordance with the methods of the invention. Exemplary antigens includebut are not limited to bacterial, viral, parasitic, allergens,autoantigens and tumor-associated antigens. If a DNA based vaccine isused the antigen will typically be encoded by a sequence of theadministered DNA construct. Alternatively, if the antigen isadministered as a conjugate the antigen will typically be a proteincomprised in the administered conjugate. Particularly, the antigen caninclude protein antigens, peptides, whole inactivated organisms, and thelike.

Specific examples of antigens that can be used include, but are notlimited to, antigens from hepatitis A, B, C or D, influenza virus,Listeria, Clostridium botulinum, tuberculosis, tularemia, Variola major(smallpox), viral hemorrhagic fevers, Yersinia pestis (plague), HIV,herpes, pappilloma virus, and other antigens associated with infectiousagents. Other antigens include antigens associated with autoimmuneconditions, inflammatory conditions, allergy, asthma and transplantrejection. Non-limiting examples of autoimmune diseases and inflammatorydiseases are provided, infra. As noted above, a B10 cell populationsensitized with a disease-specific antigen can be administered alone orin conjunction with other therapeutic agents, such as a CD40 agonist orTLR, in particular, a CD40 antibody, for use as a therapeutic orprophylactic vaccine for treating a disease condition or for suppressingimmunity. Similarly, an expanded B cell composition sensitized with adisease or microbe specific antigen may confer immunity against suchdisease conditions or microbes.

In another embodiment, a B10 cell subset may be sensitized with antigenfrom a prospective transplant donor, so as to increase the levels ofIL-10 production by the B10 cells in a transplant recipient. Theincreased IL-10 production by the B10 cell subset in the transplantrecipient results in a decreased immune/inflammatory response to thetransplant in the transplant recipient. The role of B10 cells intransplants is described more fully below.

Methods of Using the Expanded B Cell Compositions

The expanded B cell compositions may be used in methods of treatingsubjects having a disease or condition. Such adoptive transfer of Bcells, and in particular B10 cells, can be effective to suppress a widevariety of diseases, including, but not limited to autoimmune diseases,inflammatory diseases, or any other disease which may be treated byintroduction of a B cell or B10 cell population into a subject. Adoptivetransfer of B cells or B10 cells can further be employed to minimize theimmune/inflammatory response associated with transplant of cells and/ortissues.

In an exemplary adoptive transfer protocol, a mixed population of cellsis initially extracted from a target donor. Suitably, the B cells areselected, more suitably the B10 and B10pro cells are selected from thesubject. The cells isolated from the donor may be isolated from anylocation in the donor in which they reside including, but not limitedto, the blood, spleen, lymph nodes, and/or bone marrow of the donor asdescribed more fully above. Depending on the application, the cells maybe extracted from a healthy donor; a donor suffering from a disease thatis in a period of remission or during active disease; or from theorgans, blood, or tissues of a donor that has died. In the case of thelatter, the donor is an organ donor. In yet another embodiment, thecells can be obtained from the subject, expanded and/or activated andreturned to the subject.

Harvested lymphocytes may be separated by flow cytometry or other cellseparation techniques based on B cell markers or B10 cell specific cellmarkers such as those described previously (e.g., CD1d, CD5, CD24, andCD27), and then transfused to a recipient. See also InternationalApplication Nos. PCT/US2009/002560, PCT/US2011/46643 andPCT/US2011/066487, each of which is incorporated herein by reference inits entirety. Alternatively, the cells may be stored for future use. Inone aspect of this embodiment, the donor and the recipient are the samesubject. In another aspect of this embodiment, the donor is a subjectother than the recipient. In a further aspect of this embodiment, the“donor” comprises multiple donors other than the recipient, wherein theB10 cells from said multiple donors are pooled. As discussed above, theB cells obtained from the donor are expanded using the methods providedherein. The cells may also be enriched, or made to produce elevatedlevels of IL-10 by methods available to those of skill in the art priorto being administered to a recipient.

In the methods of using the expanded B cells contemplated herein,wherein the donor is a subject other than the recipient, the recipientand the donor are histocompatible. Histocompatibility is the property ofhaving the same, or mostly the same, alleles of a set of genes calledthe major histocompatibility complex (MHC). These genes are expressed inmost tissues as antigens. When transplanted cells and/or tissues arerejected by a recipient, the bulk of the immune system response isinitiated through the MHC proteins. MHC proteins are involved in thepresentation of foreign antigens to T cells, and receptors on thesurface of the T cell are uniquely suited to recognition of proteins ofthis type. MHC are highly variable between individuals, and thereforethe T cells from the host may recognize the foreign MHC with a very highfrequency leading to powerful immune responses that cause rejection oftransplanted tissue. When the recipient and the donor arehistocompatible, the chance of rejection of the B10 cell population bythe recipient is minimized.

The amount of B cells or B10 cells which will be effective in thetreatment and/or suppression of a disease or disorder which may betreated by introduction of a B cell or B10 cell population into asubject can be determined by standard clinical techniques. The dosagewill depend on the type of disease to be treated, the severity andcourse of the disease, the composition being administered, the purposeof introducing the B cell or B10 cell population, previous therapy therecipient has undertaken, the recipient's clinical history and currentcondition, and the discretion of the attending physician. For example,the specific dose for a particular subject depends on age, body weight,general state of health, diet, the timing and mode of administration,the rate of excretion, medicaments used in combination and the severityof the particular disorder to which the therapy is applied. Dosages fora given patient can be determined using conventional considerations,e.g., by customary comparison of the differential activities of thecompositions of the invention, such as by means of an appropriateconventional pharmacological or prophylactic protocol. The number ofcells administered in the composition may also be determinedempirically.

The B10 cell population can be administered in treatment regimesconsistent with the disease, e.g., a single or a few doses over one toseveral days to ameliorate a disease state or periodic doses over anextended time to inhibit disease progression and prevent diseaserecurrence. For example, the composition may be administered two or moretimes separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days,three days, four days, one week, two weeks, or by three or more weeks.The precise dose to be employed in the formulation will also depend onthe route of administration, the seriousness of the disease or disorder,and whether the disease is chronic in nature and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances.

The maximal dosage for a subject is the highest dosage that does notcause undesirable or intolerable side effects. The number of variablesin regard to an individual prophylactic or treatment regimen is large,and a considerable range of doses is expected. The route ofadministration will also impact the dosage requirements. It isanticipated that dosages of the compositions will reduce symptoms of thecondition at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%compared to pre-treatment symptoms or symptoms is left untreated. It isspecifically contemplated that pharmaceutical preparations andcompositions may palliate or alleviate symptoms of the disease withoutproviding a cure, or, in some embodiments, may be used to cure thedisease or disorder. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.Exemplary, non-limiting doses that could be used in the treatment ofhuman subjects range from at least 4×10⁴, at least 4×10⁵, at least4×10⁶, at least 4×10⁷, at least 4×10⁸, at least 4×10⁹, or at least4×10¹⁰ B cells/m². In a certain embodiment, the dose used in thetreatment of human subjects ranges from about 4×10⁸ to about 4×10¹⁰ Bcells/m².

For use in the methods described herein, the compositions may beadministered by any means known to those skilled in the art, including,but not limited to, intraperitoneal, parenteral, intravenous,intramuscular, subcutaneous, or intrathecal. Thus the compositions maysuitably be formulated as an injectable formulation. Administration ofthe compositions to a subject in accordance with the invention appearsto exhibit beneficial effects in a dose-dependent manner. Thus, withinbroad limits, administration of larger quantities of the compositions isexpected to achieve increased beneficial biological effects thanadministration of a smaller amount. Moreover, efficacy is alsocontemplated at dosages below the level at which toxicity is seen.

In another aspect, the B cells or B10 cells obtained from the donor canbe introduced into a recipient at a desired location, so as tospecifically target the therapeutic effects of the B cell or B10 cellpopulation, i.e., IL-10 production or antibody secretion. Suchtechniques can be accomplished using implantable immune modulationdevices, e.g., virtual lymph nodes, such as those described in U.S.patent application publication No. 2003/0118630; WO1999/044583; and U.S.Pat. No. 6,645,500, which are incorporated by reference herein in theirentireties. According to this embodiment, a B cell or B10 cellpopulation can be isolated from a donor as described above, added to animplantable immune modulation device, and said device then can beimplanted into a recipient at a location where the therapeutic effectsof the B cell or B10 cell population, i.e., antibodies or IL-10production, are needed.

By the terms “treat,” “treating” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is an inhibition or delay inthe progression of the condition and/or prevention or delay of the onsetof a disease or illness. The terms “treat,” “treating” or “treatment of”also means managing an autoimmune disease or disorder. Thus, the terms“treat,” “treating” or “treatment of” (or grammatically equivalentterms) refer to both prophylactic and therapeutic treatment regimes.

As used herein, a “sufficient amount” or “an amount sufficient to”achieve a particular result refers to a number of B10 or B10 effectorcells of the invention that is effective to produce a desired effect,which is optionally a therapeutic effect (i.e., by administration of atherapeutically effective amount). For example, a “sufficient amount” or“an amount sufficient to” can be an amount that is effective to alterthe severity of the subject's condition.

A “therapeutically effective” amount as used herein is an amount thatprovides some improvement or benefit to the subject. Alternativelystated, a “therapeutically effective” amount is an amount that providessome alleviation, mitigation and/or decrease in at least one clinicalsymptom. Clinical symptoms associated with the disorders that can betreated by the methods of the invention are well-known to those skilledin the art. Further, those skilled in the art will appreciate that thetherapeutic effects need not be complete or curative, as long as somebenefit is provided to the subject. It is likely that the“therapeutically effective” number of cells required to “treat” anindividual will depend on the source of the B cells, the immunologicalstatus of the patient at time of blood harvest, the condition of theindividual at the time of treatment, and the level of therapeutictreatment with immunosuppressive drugs or agents at the time oftreatment as well-known to those skilled in the art.

Specific Diseases or Conditions Treatable in the Methods

Autoimmune Diseases

Diseases and conditions associated with diminished IL-10 levels can betreated in accordance with this aspect of the invention. Decreasedlevels of IL-10 have been demonstrated in autoimmune and inflammatorydiseases including, but not limited to psoriasis (Asadullah et al.,1998, J. Clin. Investig. 101:783-94, Nickoloff et al., 1994, Clin.Immunol. Immunopathol., 73:63-8, Mussi et al. 1994, J. Biol. Regul.Homeostatic Agents), rheumatoid arthritis (Jenkins et al., 1994,Lymphokine Cytokine Res. 13:47-54; Cush et al., 1995, Arthritis Rheum.38:96-104; Al Janadi et al., 1996, J. Clin. Immunol. 16:198-207),allergic contact dermatitis (Kondo et al., 1994, J. Investig. Dermatol.103:811-14; Schwarz et al., 1994, J. Investig. Dermatol. 103:211-16),inflammatory bowel disease (Kuhn et al., 1993, Cell 75:263-74; Lindsayand Hodgson, 2001, Aliment. Pharmacol. Ther. 15:1709-16) and multiplesclerosis (Barrat et al., 2002, J. Exp. Med. 195:603-16; Cua et al.,2001, J. Immunol. 166:602-8; Massey et al., 2002, Vet. Immunol.Immunopathol. 87:357-72; Link and Xiao, 2001, Immunol. Rev. 184:117-28).

Any type of autoimmune disease can be treated in accordance with thismethod of the invention. The term “autoimmune disease or disorder”refers to a condition in a subject characterized by cellular, tissueand/or organ injury caused by an immunologic reaction of the subject toits own cells, tissues and/or organs. The term “inflammatory disease” isused interchangeably with the term “inflammatory disorder” to refer to acondition in a subject characterized by inflammation, preferably chronicinflammation. Autoimmune disorders may or may not be associated withinflammation. Moreover, inflammation may or may not be caused by anautoimmune disorder. Thus, certain disorders may be characterized asboth autoimmune and inflammatory disorders.

Exemplary autoimmune diseases or disorders include, but are not limitedto: allergic contact dermatitis, allergic reactions to drugs, alopeciaareata, ankylosing spondylitis, antiphospholipid syndrome, autoimmuneAddison's disease, autoimmune diseases of the adrenal gland, autoimmunehemolytic anemia, autoimmune hepatitis, autoimmune oophoritis andorchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid and associated skin diseases, cardiomyopathy, Celiac disease,Celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, cutaneous necrotizing venulitis,discoid lupus, erythema multiforme, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic/autoimmune thrombocytopenia purpura (ITP), immunologic lungdisease, immunologic renal disease, IgA neuropathy, juvenile arthritis,lichen planus, lupus erthematosus, Meniere's disease, mixed connectivetissue disease, multiple sclerosis, type 1 or immune-mediated diabetesmellitus, myasthenia gravis, pemphigus-related disorders (e.g.,pemphigus vulgaris), pernicious anemia, polyarteritis nodosa,polychrondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld'sphenomenon, Reiter's syndrome, Rheumatoid arthritis, rheumatic fever,sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome,spondyloarthropathies, systemic lupus erythematosis (SLE), lupuserythematosus, systemic vasculitis, takayasu arteritis, temporalarteristis/giant cell arteritis, thrombocytopenia, thyroiditis,ulcerative colitis, uveitis, vasculitides such as dermatitisherpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

The diagnosis of an autoimmune disease or disorder is complicated inthat each type of autoimmune disease or disorder manifests differentlyamong patients. This heterogeneity of symptoms means that multiplefactors are typically used to arrive at a clinical diagnosis. Generally,clinicians use factors, such as, but not limited to, the presence ofautoantibodies, elevated cytokine levels, specific organ dysfunction,skin rashes, joint swelling, pain, bone remodeling, and/or loss ofmovement as primary indicators of an autoimmune disease or disorder. Forcertain autoimmune diseases or disorders, such as RA and SLE, standardsfor diagnosis are known in the art. For certain autoimmune diseases ordisorders, stages of disease have been characterized and are well knownin the art. These art recognized methods for diagnosing autoimmunediseases and disorders as well as stages of disease and scales ofactivity and/or severity of disease that are well known in the art canbe used to identify patients and patient populations in need oftreatment for an autoimmune disease or disorder using the compositionsand methods described herein.

Diagnostic criteria for different autoimmune diseases or disorders areknown in the art. Historically, diagnosis is typically based on acombination of physical symptoms. More recently, molecular techniquessuch as gene-expression profiling have been applied to develop moleculardefinitions of autoimmune diseases or disorders. Exemplary methods forclinical diagnosis of particular autoimmune diseases or disorders areprovided below. Other suitable methods will be apparent to those skilledin the art. Also provided are methods of diagnosing and/or staging anautoimmune disease based on B10 cell numbers or activity in a subject.

In certain embodiments of the invention, patients with low levels ofautoimmune disease activity or patients with an early stage of anautoimmune disease (for diseases where stages are recognized) can beidentified for treatment using the B10 cell compositions and methodsprovided herein. The early diagnosis of autoimmune diseases is difficultdue to the general symptoms and overlap of symptoms among diseases. Insuch embodiments, a patient treated at an early stage or with low levelsof an autoimmune disease activity has symptoms comprising at least onesymptom of an autoimmune disease or disorder. In related embodiments, apatient treated at an early stage or with low levels of an autoimmunedisease has symptoms comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 symptoms of an autoimmune disease or disorder. Thesymptoms may be of any autoimmune diseases and disorders or acombination thereof. Examples of autoimmune disease and disordersymptoms are described below.

Rheumatoid Arthritis

Rheumatoid arthritis is a chronic disease, mainly characterized byinflammation of the lining, or synovium, of the joints. It can lead tolong-term joint damage, resulting in chronic pain, loss of function anddisability. Identifying patients or patient populations in need oftreatment for rheumatoid arthritis is a process. There is no definitivetest that provides a positive or negative diagnosis of rheumatoidarthritis. Clinicians rely on a number of tools including, medicalhistories, physical exams, lab tests, and X-rays.

Physical symptoms vary widely among patients and commonly include, butare not limited to, joint swelling, joint tenderness, loss of motion injoints, joint malalignment, fatigue, stiffness (particularly in themorning and when sitting for long periods of time), weakness, flu-likesymptoms (including a low-grade fever), pain associated with prolongedsitting, the occurrence of flares of disease activity followed byremission or disease inactivity, rheumatoid nodules or lumps of tissueunder the skin (typically found on the elbows, they can indicate moresevere disease activity), muscle pain, loss of appetite, depression,weight loss, anemia, cold and/or sweaty hands and feet, and involvementof the glands around the eyes and mouth, causing decreased production oftears and saliva (Sjögren's syndrome). Apart from physical symptoms,clinicians commonly use tests, such as, but not limited to, completeblood count, erythrocyte sedimentation rate (ESR or sed rate),C-reactive protein, rheumatoid factor, antinuclear antibodies (ANA),imaging studies, radiographs (X-rays), magnetic resonance imaging (MM)of joints or organs, joint ultrasound, and bone densitometry (DEXA).These tests are examples of tests that can be used in conjunction withthe compositions and methods described herein to check for abnormalitiesthat might exist (i.e., identify patients or patient populations in needof treatment) or to monitor side effects of drugs and check progress.

Early symptoms of rheumatoid arthritis commonly are found in the smallerjoints of the fingers, hands and wrists. Joint involvement is usuallysymmetrical, meaning that if a joint hurts on the left hand, the samejoint will hurt on the right hand. In general, more joint erosionindicates more severe disease activity.

Symptoms of more advanced disease activity include damage to cartilage,tendons, ligaments and bone, which causes deformity and instability inthe joints. The damage can lead to limited range of motion, resulting indaily tasks (grasping a fork, combing hair, buttoning a shirt) becomingmore difficult. Skin ulcers, greater susceptibility to infection, and ageneral decline in health are also indicators of more advanced diseaseactivity.

Progression of rheumatoid arthritis is commonly divided into threestages. The first stage is the swelling of the synovial lining, causingpain, warmth, stiffness, redness and swelling around the joint. Secondis the rapid division and growth of cells, or pannus, which causes thesynovium to thicken. In the third stage, the inflamed cells releaseenzymes that may digest bone and cartilage, often causing the involvedjoint to lose its shape and alignment, more pain, and loss of movement.

Molecular techniques can also be used to to identify patients or patientpopulations in need of treatment. For example, rheumatoid arthritis hasbeen shown to be associated with allelic polymorphisms of the humanleukocyte antigen (HLA)-DR4 and HLA-DRB1 genes. Rheumatoid arthritispatients frequently express two disease-associated HLA-DRB1*04 alleles.Patients can be tested for allelic polymorphisms using methods standardin the art. MHC genes are not the only germline-encoded genesinfluencing susceptibility to RA that can be used to diagnose oridentify patients or patient populations in need of treatment. Femalesex clearly increases the risk, and female patients develop a differentphenotype of the disease than do male patients. Any molecular indicatorsof rheumatoid arthritis can be used to identify patients or patientpopulations in need of treatment with B10 cell compositions and methodsdescribed herein.

In certain embodiments of the methods, a patient can be treated with B10cell compositions prior, concurrent, or subsequent to other therapies,such as, but not limited to surgery. For example, patients in need oftreatment for rheumatoid arthritis commonly undergo surgical treatment,such as, but not limited to, synovectomy to reduce the amount ofinflammatory tissue by removing the diseased synovium or lining of thejoint; arthroscopic surgery to take tissue samples, remove loosecartilage, repair tears, smooth a rough surface or remove diseasedsynovial tissue; osteotomy, meaning “to cut bone,” this procedure isused to increase stability by redistributing the weight on the joint;joint replacement surgery or arthroplasty for the surgicalreconstruction or replacement of a joint; or arthrodesis or fusion tofuse two bones together. Such surgical procedures are examples oftreatment that patients can undergo prior, concurrent, or subsequent totreatment with the methods and compositions provided herein. In oneembodiment, the B10 cell compositions may be administered locally at thesite of surgery either before, during or after surgery to protect thejoint from further attack or degradation.

Systemic Lupus Erythematosis (SLE)

Systemic lupus erythematosis (SLE) is a chronic (long-lasting) rheumaticdisease which affects joints, muscles and other parts of the body.Patients or patient populations in need of treatment for SLE can beidentified by examining physical symptoms and/or laboraotry testresults. Physical symptoms vary widely among patients. For example, inSLE, typically 4 of the following 11 symptoms exist before a patient isdiagnosed with SLE: 1) malar rash: rash over the cheeks; 2) discoidrash: red raised patches; 3) photosensitivity: reaction to sunlight,resulting in the development of or increase in skin rash; 4) oralulcers: ulcers in the nose or mouth, usually painless; 5) arthritis:nonerosive arthritis involving two or more peripheral joints (arthritisin which the bones around the joints do not become destroyed); 6)serositis pleuritis or pericarditis: (inflammation of the lining of thelung or heart); 7) renal disorder: excessive protein in the urine(greater than 0.5 gm/day or 3+ on test sticks) and/or cellular casts(abnormal elements the urine, derived from red and/or white cells and/orkidney tubule cells); 8) neurologic disorder: seizures (convulsions)and/or psychosis in the absence of drugs or metabolic disturbances whichare known to cause such effects; 9) hematologic disorder: hemolyticanemia or leukopenia (white blood count below 4,000 cells per cubicmillimeter) or lymphopenia (less than 1,500 lymphocytes per cubicmillimeter) or thrombocytopenia (less than 100,000 platelets per cubicmillimeter) (The leukopenia and lymphopenia must be detected on two ormore occasions. The thrombocytopenia must be detected in the absence ofdrugs known to induce it); 10) antinuclear antibody: positive test forantinuclear antibodies (ana) in the absence of drugs known to induce it;and/or 11) immunologic disorder: positive anti-double stranded anti-DNAtest, positive anti-sm test, positive antiphospholipid antibody such asanticardiolipin, or false positive syphilis test (vdrl).

Other physical symptoms that may be indicative of SLE include, but arenot limited to, anemia, fatigue, fever, skin rash, muscle aches, nausea,vomiting and diarrhea, swollen glands, lack of appetite, sensitivity tocold (Raynaud's phenomenon), and weight loss.

Laboratory tests can also be used to to identify patients or patientpopulations in need of treatment. For example, a blood test can be usedto detect a group of autoantibodies found in the blood of almost allpeople with SLE; a compliment test (C3, C4, CH50, CH100) can be used tomeasure the amount of complementary proteins circulating in the blood, asedimentation rate (ESR) or C-reactive protein (CRP) may be used tomeasure inflammation levels, a urine analysis can be used to detectkidney problems, chest X-rays may be taken to detect lung damage, and anEKG can be used to detect heart problems. These tests are examples oftests that can be used in conjunction with the compositions and methodsdescribed herein to check for abnormalities that might exist (i.e.,identify patients or patient populations in need of treatment) or tomonitor side effects of drugs and check progress.

Idiopathic/Autoimmune Thrombocytopenia Purpura (ITP)

Idiopathic/autoimmune thrombocytopenia purpura (ITP) is a disorder ofthe blood characterized by immunoglobulin G (IgG) autoantibodies thatinteract with platelet cells resulting in destruction of those plateletcells. Typically, the antibodies are specific to platelet membraneglycoproteins. The disorder may be acute (temporary, lasting less than 2months) or chronic (persisting for longer than 6 months). Patients orpatient populations in need of treatment for ITP can be identified byexamining a patient's medical history, physical symptoms, and/orlaboratory test results.

Physical symptoms include purplish-looking areas of the skin and mucousmembranes (such as the lining of the mouth) where bleeding has occurredas a result of a decrease in the number of platelet cells. The mainsymptom is bleeding, which can include bruising (“ecchymosis”) and tinyred dots on the skin or mucous membranes (“petechiae”). In someinstances bleeding from the nose, gums, digestive or urinary tracts mayalso occur. Rarely, bleeding within the brain occurs. Common signs,symptoms, and precipitating factors also include, but are not limitedto, abrupt onset (childhood ITP), gradual onset (adult ITP), nonpalpablepetechiae, purpura, menorrhagia, epistaxis, gingival bleeding,hemorrhagic bullae on mucous membranes, signs of GI bleeding,menometrorrhagia, evidence of intracranial hemorrhage, nonpalpablespleen, retinal hemorrhages, recent live virus immunization (childhoodITP), recent viral illness (childhood ITP), spontaneous bleeding whenplatelet count is less than 20,000/mm³, and bruising tendency.

Laboratory tests that can be used to diagnose ITP include, but are notlimited to, a complete blood count test, or a bone marrow examination toverify that there are adequate platelet-forming cells (megakaryocyte) inthe marrow and to rule out other diseases such as metastatic cancer andleukemia. Isolated thrombocytopenia is the key finding regardinglaboratory evaluation. Giant platelets on peripheral smear areindicative of congenital thrombocytopenia. A CT scan of the head may bewarranted if concern exists regarding intracranial hemorrhage. Thesetests are examples of tests that can be used in conjunction with thecompositions and methods described herein to check for abnormalitiesthat might exist (i.e., identify patients or patient populations in needof treatment) or to monitor side effects of drugs and check progress.

Pemphigus-Related Disorders

Pemphigus-related disorders are characterized by a blistering conditionof the skin caused by the attack of antibodies of certain proteins onthe surface of skin cells. This attack interferes with the ability ofthe skin cells to bind to each other. There are three main types ofpemphigus: pemphigus vulgaris, pemphigus foliaceus and paraneoplasticpemphigus. Patients or patient populations in need of treatment forpemphigus-related disorders can be identified by examining a patient'smedical history, physical symptoms, and/or laboratory test results.

Typically, diagnosis of these pemphigus-related disorders is made byskin biopsy. The biopsy skin sample can be treated using a directimmunoflourescence technique to detect desmoglein antibodies in theskin. Another diagnostic test that may be used is called indirectimmunofluorescence or antibody titer test. This measures desmogleinautoantibodies in the blood serum. It may be used to obtain a morecomplete understanding of the course of the disease. In addition, aserum assay for desmoglein antibodies, an ELISA, is also available. Itis the most accurate. The presence of these desmoglein autoantibodies inbiopsy samples is diagnostic of pemphigus generally.

Pemphigus vulgaris can be diagnosed by the presence of blisters in themouth. Inflammation or erosions may also be present in the lining of theeye and eyelids, and the membranes of the nose or genital tract. Half ofthe patients also develop blisters or erosions of the skin, often in thegroin, underarm, face, scalp and chest areas. Pemphigus foliaceus is asuperficial, relatively mild form of pemphigus. It usually manifests onthe face and scalp, but also involves the back and chest. Lesions do notoccur in the mouth. The blisters are more confined to the outermostsurface and often itch. Paraneoplastic pemphigus is very rare andgenerally occurs in people who have cancer. The lesions are painful andaffect the mouth, lips and esophagus (swallowing tube) as well as theskin. Due to involvement of the airways, signs of respiratory diseasemay occur and can be life-threatening. These tests are examples of teststhat can be used in conjunction with the compositions and methodsdescribed herein to check for abnormalities that might exist (i.e.,identify patients or patient populations in need of treatment) or tomonitor side effects of drugs and check progress.

Autoimmune Diabetes

A patient in need of treatment for type 1 diabetes can be treated withthe B10 cell compositions and methods described herein as well. Type 1diabetes is an autoimmune disease, in which the body's immune systemdamages the islet cells in the pancreas, reducing the production ofinsulin, resulting in high blood sugar. Patients or patient populationsin need of treatment for autoimmune diabetes can be identified byexamining a patient's medical history, physical symptoms, and/orlaboratory test results. Symptoms often come on suddenly and include,but are not limited to, increased thirst, increased urination, constanthunger, weight loss, blurred vision, and fatigue.

Laboratory tests that can be used in identifying patients or patientpopulations in need of treatment for autoimmune diabetes include, butare not limited to, the direct measurement of glucose levels in theblood during an overnight fast, and measurement of the body's ability toappropriately handle the excess sugar presented after drinking a highglucose drink. For the first test, an elevated blood sugar level afteran overnight fast (not eating anything after midnight) can be used as adiagnostic factor. A value above 140 mg/dl on at least two occasionstypically means a patient has diabetes. Normal patients have fastingsugar levels that generally run between 70-110 mg/dl. For the secondtest, an oral glucose tolerance test is typically performed. The patientbeing tested starts the test in a fasting state (having no food or drinkexcept water for at least 10 hours but not greater than 16 hours). Aninitial blood sugar is drawn and then the patient is given a “glucola”bottle with a high amount of sugar in it (75 grams of glucose), (or 100grams for pregnant women). The patient then has their blood tested again30 minutes, 1 hour, 2 hours and 3 hours after drinking the high glucosedrink. These tests are examples of tests that can be used in conjunctionwith the compositions and methods described herein to check forabnormalities that might exist (i.e., identify patients or patientpopulations in need of treatment) or to monitor side effects of drugsand check progress.

Scleroderma

Scleroderma is a chronic skin and connective tissue disease. In general,it is characterized by the formation of scar tissue in the skin andorgans of the body. Identification of patients and patient populationsin need of treatment of scleroderma can be based on clinical history andphysical findings. Patients or patient populations in need of treatmentfor scleroderma can be identified by examining a patient's medicalhistory, physical symptoms, and/or laboraotry test results. Diagnosismay be delayed in patients without significant skin thickening.Laboratory, X-ray, pulmonary function tests, and skin or renal (kidney)biopsies can be used to determine the extent and severity of internalorgan involvement.

There are two types of scleroderma. Localized scleroderma affects theskin in limited areas and the musculoskeletal system. Systemic sclerosiscauses more widespread skin changes and may be associated with internalorgan damage in the lungs, heart and kidneys. Scleroderma sharessymptoms that are common with other autoimmune diseases, including butnot limited to, numbness, pain or color changes in fingers, toes,cheeks, nose and ears, often brought on by cold or emotional distress(Raynaud's phenomenon), stiffness or pain in your joints and curling ofyour fingers, digestive problems ranging from poor absorption ofnutrients to delayed movement of food due to impaired muscular activityin your intestine, sores over joints, such as your elbows and knuckles,and puffy hands and feet, particularly in the morning. It can alsoinvolve arthritis, muscle inflammation, dry eyes and dry mouth. Mostpatients with scleroderma have cold-induced spasms of small bloodvessels in their hands or feet, known as Raynaud's phenomenon, whichcauses the fingers or toes to turn white or blue and may be painful. Thesymptoms of scleroderma vary greatly from individual to individual, andthe effects of scleroderma can range from very mild to life-threatening.

Localized scleroderma has three main subtypes, which are called morphea,generalized morphea and linear scleroderma. Systemic type is the moreserious type because it affects internal organ systems. Its threesubtypes are called limited, diffuse and sine.

In the early months or years of disease onset, scleroderma may resemblemany other connective tissue diseases, such as, but not limited to,Systemic Lupus Erythematosus, Polymyositis, and Rheumatoid Arthritis.

The most classic symptom of systemic sclerosis (scleroderma) issclerodactyly. Initial symptoms include swollen hands, which sometimesprogress to this tapering and claw-like deformity. Not everyone withscleroderma develops this degree of skin hardening. Other symptoms caninclude morphea, linear sclerodactyly (hardened fingers), Raynaud'ssyndrome, calcinosis, and telangiectasia.

Blood tests such as antinuclear antibody (ANA) tests can be used in thediagnosis of both localized and systemic scleroderma. For example,anti-centromere antibodies (ACA) and anti-Scl-70 antibodies areindicative of patients in need of treatment for systemic sclerosis (Hoet al., 2003, Arthritis Res Ther. 5:80-93); anti-topo II alpha antibodyis indicative of patients in need of treatment for local scleroderma;and anti-topo I alpha antibody is indicative of patients in need oftreatment for systemic scleroderma.

Several types of scleroderma and methods for diagnosing these types arerecognized and well known in the art, including, but not limited to,juvenile scleroderma; localized scleroderma; Nodular Scleroderma; andSystemic scleroderma, including, but not limited to, Calcinosis,Raynaud's, Esophagus, Sclerodactyly, and Telangiectasia (CREST), limitedsystemic scleroderma, and diffuse systemic scleroderma. Systemicscleroderma is also known as systemic sclerosis (SSc). It may also bereferred to as Progressive Systemic Sclerosis (PSSc), or FamilialProgressive Systemic Sclerosis (FPSSc). Systemic sclerosis is amultisystem disorder characterized by the presence of connective tissuesclerosis, vascular abnormalities concerning small-sized arteries andthe microcirculation, and autoimmune changes.

The type of systemic scleroderma known as CREST is not characterized byany skin tightening. CREST is characterized by Calcinosis (calciumdeposits), usually in the fingers; Raynaud's; loss of muscle control ofthe Esophagus, which can cause difficulty swallowing; Sclerodactyly, atapering deformity of the bones of the fingers; and Telangiectasia,small red spots on the skin of the fingers, face, or inside of themouth. Typically two of these symptoms is sufficient for diagnosis ofCREST. CREST may occur alone, or in combination with any other form ofScleroderma or with other autoimmune diseases.

Limited Scleroderma is characterized by tight skin limited to thefingers, along with either pitting digital ulcers (secondary toRaynaud's) and/or lung fibrosis. The skin of the face and neck may alsobe involved in limited scleroderma.

Diffuse Scleroderma is diagnosed whenever there is proximal tight skin.Proximal means located closest to the reference point. Proximal tightskin can be skin tightness above the wrists or above the elbows.Typically, a patient with skin tightness only between their elbows andtheir wrists will receive a diagnosis of either diffuse or limitedsystemic Scleroderma, depending on which meaning of proximal thediagnosing clinician uses. These tests are examples of tests that can beused in conjunction with the compositions and methods described hereinto check for abnormalities that might exist (i.e., identify patients orpatient populations in need of treatment) or to monitor side effects ofdrugs and check progress.

Activity of Autoimmune Diseases or Disorders

According to certain aspects, the patient or patient population beingtreated with B10 cell compositions described herein can have anautoimmune disease or disorder with a certain activity. The activity ofan autoimmune disease or disorder can be measured by assessing multiplefactors, such as, but not limited to those described above for diagnosisof autoimmune diseases and disorders. Although the above-describedfactors are presented in the context of certain autoimmune diseases, oneor more of these factors can be used to determine the activity of otherautoimmune diseases or disorders. Methods for determining activity of anautoimmune disease or disorder in a patient in relation to a scale ofactivity are well known in the art and can be used in connection withthe pharmaceutical compositions and methods described herein.

For example, the American College of Rheumatologists Score (ACR score)can be used to determine the activity of rheumatoid arthritis of apatient or a patient population. According to this method, patients aregiven a score that correlates to improvement. For example, patients witha 20% improvement in factors defined by the ACR would be given an ACR20score. This and other scoring methods may be used in combination withthe methods of assessing B10 cell function described herein.

For certain other autoimmune diseases or disorders, there are severalaccepted methods that can be used in conjunction with the methodsprovided here to determine activity of an autoimmune disease ordisorder. For example, SLE at several disease assessment scales,including, but not limited to, British Isle Lupus Assessment Group(BILAG), Systemic Lupus Erythematosus Disease Activity Index (SLEDI),Modified SLEDI, and Systemic Lupus Activity measure (SLAM). In general,a high activity of an autoimmune disease or disorder would be one whichscores in the upper half (e.g., greater severity of disease activity) ofone or more of the above scales and a low activity of an autoimmunedisease or disorder would be one which scores in the lower half (e.g.,less severity of disease activity) of one or more of the above scales.

Inflammatory and Allergic Diseases

Any type of inflammatory disease can be treated in accordance with themethods described herein. Non-limiting examples of inflammatory diseasesinclude, but are not limited to, asthma, encephilitis, inflammatorybowel disease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentiatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, and chronic inflammation resulting from chronicviral or bacterial infections.

The methods of the invention encompass therapies that are aimed attreating diseases associated with a helper T (Th) 1-mediatedinflammatory response but not diseases associated with a Th2-mediatedinflammatory response. In an alternative aspect of this embodiment, themethods of the invention encompass therapies that are aimed at treatingdiseases associated with a Th2-mediated inflammatory response but notdiseases associated with a Th1-mediated inflammatory response.

Transplantation

IL-10 is capable of inhibiting ischemia/reperfusion injury (Deng et al.,2001, Kidney Int. 60:2118-28), graft-versus-host disease, andtransplant-related mortality (Baker et al., 1999, Bone Marrow Transplant23:1123-9; Holler et al., 2000, Bone Marrow Transplant 25:237-41). Assuch, one embodiment of the present invention involves treatingtransplant-associated diseases/conditions by increasing the level ofIL-10 in a patient in need thereof.

In one embodiment, the levels of endogenous IL-10 are increased in asubject receiving an organ transplant by administration of a B10 cellsubset such as B10 cells made by the methods described herein. In oneaspect of this embodiment, the B10 cell population is isolated from thepatient themselves, i.e., the subject is the donor. In another aspect ofthis embodiment, the B10 cell population is isolated from a donor thatis not the subject. The donor of the B10 cells may be the same as theorgan donor. In another embodiment, the B10 cell population is pooledfrom several donors.

According to certain aspects of the invention, the treatment regimen anddose used with the compositions and methods of the invention is chosenbased on a number of factors including, for example, clinicalmanifestation that place a patient at risk for developing humoral orcellular rejection, or clinical evidence that such a rejection isdeveloping. The terms “humoral” and “antibody-mediated” are usedinterchangeably herein. The criteria for assessing the risk that apatient will develop humoral or cellular rejection are establishedaccording to the knowledge and skill in the art. In one embodiment, apositive complement dependent cytotoxicity or anti-globulin enhancedcomplement-dependent cytotoxicity crossmatch indicates that a patient isat high risk for humoral rejection. In one embodiment, a positivecrossmatch or a prior positive complement dependent cytotoxicity oranti-globulin enhanced complement dependent cytotoxicity crossmatchindicates that a patient is at an intermediate risk for humoralrejection. In one embodiment, a negative crossmatch indicates that apatient is at a low risk for humoral rejection. Similarly, there areestablished criteria known to those of skill in the art for establishingrisk for cellular transplant rejection.

In another embodiment, a transplant recipient in need of prophylaxisagainst graft rejection may be identified as a patient or patientpopulation having detectable circulating anti-HLA alloantibodies priorto transplantation. In another example, the patient or patientpopulation is identified as having panel reactive antibodies prior totransplantation. The presence of detectable circulating anti-HLAalloantibodies in a transplant recipient post-transplantation can alsobe used to identify the patient or patient population in need oftreatment for humoral or cellular rejection according to the invention.The patient or patient population in need of treatment for humoral orcellular rejection can also be identified according to other clinicalcriteria that indicate that a transplant recipient is at risk fordeveloping humoral or cellular rejection or has already developedhumoral or cellular rejection. For example, a transplant recipient inneed of treatment of rejection may be identified as a patient orpopulation in an early stage of humoral rejection, such as a latenthumoral response characterized by circulating anti-donor alloantibodies.An early stage of humoral rejection may also be a silent reactioncharacterized by circulating anti-donor alloantibodies and C4ddeposition, or a subclinical rejection characterized by circulatinganti-donor alloantibodies, C4d deposition, and tissue pathology. Inlater stages, the recipient is identified as a patient or patientpopulation presenting with clinical indications of humoral or cellularrejection characterized according to the knowledge and skill in the art,for example, by circulating anti-donor alloantibodies, C4d deposition,tissue pathology and/or inflammatory cell infiltrates, and graftdysfunction.

The present invention provides compositions, therapeutic formulations,methods and regimens effective to reduce the incidence, severity, orduration of graft-versus-host disease (GVHD), or a humoral or cellularrejection episode. In certain embodiments, the compositions and methodsof the invention are effective to attenuate the host response toischemic repefusion injury of a solid tissue or organ graft. In apreferred embodiment, the B10 effector cell compositions and methods ofthe invention are effective to prolong survival of a graft in atransplant recipient.

The present invention encompasses grafts that are autologous,allogeneic, or xenogeneic to the recipient. The types of graftsencompassed by the invention include tissue and organ grafts, includingbut not limited to, bone marrow grafts, peripheral stem cell grafts,skin grafts, arterial and venous grafts, pancreatic islet cell grafts,organ transplants of the kidney, liver, lung, pancreas, thyroid, andheart, and composite tissue grafts involving tissues from multiple organsystems, including but not limited to digits, limbs, regions of thebody, and facial tissues. The terms “graft” and “transplant” are usedinterchangeably herein. In one embodiment, the autologous graft is abone marrow graft, an arterial graft, a venous graft or a skin graft. Inone embodiment, the allograft is a bone marrow graft, a corneal graft, akidney transplant, a pancreatic islet cell transplant, or a combinedtransplant of a kidney and pancreas. In one embodiment, the graft is axenograft, preferably wherein the donor, is a pig. The compositions andmethods of the present invention may also be used to suppress adeleterious immune response to a non-biological graft or implant,including but not limited to an artificial joint, a stent, or apacemaker device.

The B10 effector cell compositions, and methods of the invention can beused to treat or prevent GVHD, or humoral or cellular rejection withoutregard to the particular indications initially giving rise to the needfor the transplant or the particular type of tissue transplanted.However, the indications that gave rise to the need for a transplant andthe type of tissue transplanted can provide a basis for a comprehensivetherapeutic regimen for the treatment or prevention of GVHD and graftrejection, which comprehensive regimen comprises the B10 effector cellcompositions and methods of the invention.

Therapeutic formulations and regimens of the present invention aredescribed elsewhere for treating human subjects with other conditions.Similarly, appropriate treatment regimens can be determined by one ofskill in the art for the particular patient or patient population. Inparticular embodiments, the treatment regimen is a pre-transplantconditioning regimen, a post-transplant maintenance regimen, orpost-transplant treatment regimen for acute or chronic rejection. Incertain embodiments, the particular regimen is varied for a patient whois assessed as being at a high or intermediate risk of developing ahumoral or cellular immune response, compared with the regimen for apatient who is assessed as being at a low risk of developing a humoralor cellular response directed against the transplant.

In certain embodiments, the particular regimen is varied according tothe stage of transplant rejection, with more aggressive therapy beingindicated for patients at later stages of humoral or cellular rejection.The stages of humoral rejection may be classified according to theknowledge and skill in the art. For example, the stages of humoralrejection may be classified as one of stages I to IV according to thefollowing criteria: Stage I Latent Response, characterized bycirculating anti-donor alloantibodies, especially anti-HLA antibodies;Stage II Silent Reaction, characterized by circulating anti-donoralloantibodies, especially anti-HLA antibodies, and C4d deposition, butwithout histologic changes or graft dysfunction; Stage III SubclinicalRejection: characterized by circulating anti-donor alloantibodies,especially anti-HLA antibodies, C4d deposition, and tissue pathology,but without graft dysfunction; Stage IV Humoral Rejection: characterizedby circulating anti-donor alloantibodies, especially anti-HLAantibodies, C4d deposition, tissue pathology, and graft dysfunction.Similarly, criteria for cellular rejection are known to those practicingin the art.

Dose response curves can be generated using standard protocols in theart in order to determine the effective amount of the compositions ofthe invention for use in a particular regimen, for example, inconditioning regimens prior to transplantation, and inpost-transplantation regimens for prophylaxis and treatment of GVHD, orhumoral or cellular rejection. In general, patients at high risk fordeveloping humoral or cellular rejection and those already exhibitingone or more clinical indicators of rejection will require higher dosesand/or more frequent doses which may be administered over longer periodsof time in comparison to patients who are not at high risk or who do notexhibit any indications of active rejection.

The B10 effector cell compositions and methods of the invention can bepracticed to treat or prevent GVHD, or humoral or cellular rejection,either alone or in combination with other therapeutic agents ortreatment regimens. Other therapeutic regimens for the treatment orprevention of GVHD, or humoral or cellular rejection may comprise, forexample, one or more of anti-lymphocyte therapy, steroid therapy,antibody depletion therapy, immunosuppression therapy, andplasmapheresis. Anti-lymphocyte therapy may comprise the administrationto the transplant recipient of anti-thymocyte globulins, also referredto as thymoglobulin. Anti-lymphocyte therapy may also comprise theadministration of one or more monoclonal antibodies directed against Tcell and including B cell surface antigens. Examples of such antibodiesinclude, without limitation, OKT3™ (muromonab-CD3), CAMPATH™-1H(alemtuzumab), CAMPATHT™-1G, CAMPATH™-1M, SIMULECT™ (basiliximab), andZENAPAX™ (daclizumab).

Steroid therapy may comprise administration to the transplant recipientof one or more steroids selected from the group consisting of cortisol,prednisone, methyl prednisolone, dexamethazone, and indomethacin.Preferably, one or more of the steroids are corticosteroids, includingwithout limitation, cortisol, prednisone, and methylprednisolone.

Antibody depletion therapy may include, for example, administration tothe transplant recipient of intravenous immunoglobulin. Antibodydepletion therapy may also comprise immunoadsorption therapy applied tothe graft ex vivo, prior to transplantation. Immunoadsorption may beaccomplished using any suitable technique, for example, protein Aaffinity, or antibody based affinity techniques using antibodiesdirected against T cell or B cell surface markers such as anti-CD3antibodies.

Immunosuppression therapy may comprise the administration of one or moreimmunosuppressive agents such as inhibitors of cytokine transcription(e.g., cyclosporin A, tacrolimus), nucleotide synthesis (e.g.,azathiopurine, mycophenolate mofetil), growth factor signal transduction(e.g., sirolimus, rapamycin), and the T cell IL-2 receptor (e.g.,daclizumab, basiliximab). In a particular embodiment, animmunosuppressant agent used in combination with the compositions andmethods of the invention includes one or more of the following:adriamycin, azathiopurine, busulfan, cyclophosphamide, cyclosporin A(“CyA”), cytoxin, fludarabine, 5-fluorouracil, methotrexate,mycophenolate mofetil (MOFETIL), nonsteroidal anti-inflammatories(NSAIDs), rapamycin, and tacrolimus (FK506). Immunosuppressive agentsmay also comprise inhibitors of complement, for example, solublecomplement receptor-1, anti-05 antibody, or a small molecule inhibitorof Cls, for example as described in Buerke et al. (J. Immunol.,167:5375-80 (2001). In one embodiment, the compositions and methods ofthe invention are used in combination with one or more therapeuticregimens for suppressing humoral or cellular rejection, including,without limitation, tacrolimus and mycophenolate mofetil therapy,immunoadsorption, intravenous immunoglobulin therapy, andplasmapheresis.

Diagnosis and Clinical Criteria

The present invention provides antibodies, compositions and methods fortreating and preventing GVHD, and humoral or cellular rejection in humantransplant recipients. The compositions and methods of the invention canbe used regardless of the particular indications that gave rise to theneed for a transplant. Similarly, the use of the compositions andmethods of the invention for the treatment and prevention of GVHD, andhumoral or cellular rejection is not limited by the particular type oftissue which is intended for transplantation or which has beentransplanted.

In one embodiment, the invention provides compositions and methods forthe prevention of humoral or cellular rejection in a human transplantrecipient wherein the transplant recipient is identified as a patient orpatient population at increased risk for developing humoral or cellularrejection. Such patients may also be referred to as “sensitized.”Criteria for the identification of sensitized patients are known toskilled practitioners. Such criteria may include, for example, patientshaving detectable levels of circulating antibodies against HLA antigens,e.g., anti-HLA alloantibodies. Such criteria may also include patientswho have undergone previous transplantations, a pregnancy, or multipleblood transfusions. Patients who are at an increased risk for humoralrejection also include those having imperfect donor-recipient HLAmatching, and those transplants that are ABO-incompatible. Sensitizedindividuals are preferred candidates for pretreatment or conditioningprior to transplantation. Sensitized individuals are also preferredcandidates for post-transplantation maintenance regimens for theprevention of humoral and cellular rejection.

In one embodiment, the B10 cell compositions and methods of theinvention comprise or are used in combination with a therapeutic regimenfor the treatment of an acute or chronic rejection. In particularembodiments, the rejection is characterized as Stage I, Stage II, StageIII, or Stage IV humoral or cellular rejection.

In one embodiment, the B10 cell compositions and methods of theinvention comprise or are used in combination with a therapeutic regimenfor the treatment of an early stage humoral rejection. In particularembodiments, the early stage humoral rejection is Stage I, II, or IIIrejection. Clinical indications of an early stage humoral rejection aredetermined according to the knowledge and skill in the art and mayinclude, for example, the development in the patient of circulatingdonor-specific anti-HLA antibodies, the presence of complement markersof antibody activity such as C4d and C3d deposits in graft biopsies, andthe presence of anti-HLA antibodies in graft biopsies. Other indicatorsof an early stage humoral rejection are known to the skilledpractitioner and may include, for example, the development ofanti-endothelial antibodies, especially anti-vimentin antibodies, andthe development of nonclassical MHC class I-related chain A (MICA)alloantibodies. In one embodiment, the compositions and methods of theinvention comprise or are used in combination with a therapeutic regimenfor the treatment of humoral or cellular rejection characterized in partby graft dysfunction. In particular embodiments, the patient or patientpopulation in need of treatment for humoral or cellular rejection isidentified according to criteria known in the art for graft dysfunction.Examples of such criteria for particular types of grafts are provided inthe sections that follow. In other embodiments, the patient or patientpopulation in need of treatment for humoral or cellular rejection isidentified according to other criteria that are particular to the typeof tissue graft, such as histological criteria. Examples of suchcriteria are also provided in the sections that follow.

Bone Marrow Transplants

The compositions and methods of the invention are useful for treating orpreventing GVHD, and humoral or cellular rejection in a bone marrowtransplant recipient. In one embodiment, the compositions and methods ofthe invention comprise or are used in combination with a pre-transplantconditioning regimen. The graft may be from any suitable source, forexample, cord blood stem cells, peripheral blood stem cells, or a bonemarrow harvest. Peripheral blood stem cells may be harvested from donorblood following a suitable conditioning regimen. Suitable regimens areknown in the art and may include, for example, administration of one ormore of the following to the donor prior to harvesting the donor blood:NEUPOGEN, cytokines such as GM-CSF, low dose chemotherapeutic regimens,and chemokine therapy. The graft may be either allogeneic or autologousto the transplant recipient. The graft may also be a xenograft.

The compositions and methods of the invention are useful in a number ofcontexts in which there is a hematopoietic indication for bone marrowtransplantation. In one embodiment, an autologous bone marrow graft isindicated for a B cell leukemia or lymphoma, preferably acutelymphoblastic leukemia (“ALL”) or non-Hodgkins lymphoma. In anotherembodiment, the graft is an allogeneic graft and the B10 effector cellcompositions and methods of the invention are used for treating graftrecipients as prophylaxis against GVHD.

In one embodiment, the indication is a B cell associated autoimmunecondition and the compositions and methods of the invention are used astherapy conditioning regimens. In one embodiment, the compositions ofthe invention are administered in combination with a chemotherapy orradiation therapy regimen, which regimen comprises a lower dose of oneor more chemotherapeutic agents, or a lower dose of radiation, than thedose that is administered in the absence of the compositions of theinvention. In one embodiment, the patient receives an autologous bonemarrow graft subsequent to chemotherapy or radiation therapy, whereinthe graft recipient is subsequently treated using the compositions andmethods described herein. A patient or patient population in need of, orlikely to benefit from, a bone marrow transplant is identified accordingto the knowledge and skill in the art. Examples of patients that may becandidates for bone marrow transplantation include patients who haveundergone chemotherapy or radiation therapy for the treatment of acancer or an autoimmune disease or disorder, and patients who are unableto clear a viral infection residing in cells of the immune system.

Liver Transplants

The compositions and methods of the invention are useful for treating orpreventing GVHD, and humoral or cellular rejection in a liver transplantrecipient. In particular embodiments, the rejection is an acute or achronic refection. In one embodiment, the compositions and methods ofthe invention are used for the prevention of GVHD, and humoral orcellular rejection in a liver transplant recipient. In one embodiment,the compositions and methods of the invention comprise or are used incombination with a pre-transplant conditioning regimen. The livertransplant may be from any suitable source as determined according tothe knowledge and skill in the art. In one embodiment, the liver is anHLA-matched allogeneic graft. In another embodiment, the liver is axenograft, preferably from a pig donor. In one embodiment, the liver isused ex vivo to filter the patient's blood, e.g., extracorporealperfusion. Extracorporeal perfusion is a form of liver dialysis in whichthe patient is surgically connected to a liver maintained outside thebody. This procedure is sometimes referred to as “bioartificial liver.”In accordance with this embodiment, the compositions and methods of theinvention are used to prevent the development of antibodies andsensitized lymphocytes against liver antigens which may contaminate thepatient's blood. In one embodiment, the compositions and methods of theinvention comprise an improved therapeutic regimen for the treatment andprevention of GVHD, and humoral or cellular rejection. In a particularembodiment, the compositions and methods of the invention comprise animproved therapeutic regimen, wherein the improvement lies in adecreased incidence and/or severity of complications associated withtraditional immunosuppressive agents. In one embodiment, the incidenceand/or severity of nephrotoxicity, hepatotoxicity, and hirsutism isreduced compared with traditional regimens relying on cyclosporinA orother calcinuerin inhibitors. In one embodiment, the incidence and/orseverity of obesity, osteodystrophy, diabetes mellitus andsusceptibility to bacterial and viral infections is reduced comparedwith traditional regimens relying on corticosteroids. In a preferredembodiment, the compositions and methods of the invention are used incombination with lower doses of one or more traditionalimmunosuppressive agents than the doses that are used in the absence ofanti-lymphocyte antibody therapy. Preferably, the lower doses result ina decreased incidence and/or severity of one or more complicationsassociated with the one or more traditional immunosuppressive agents.

A patient or patient population in need of, or likely to benefit from, aliver transplant is identified according to the knowledge and skill inthe art. Examples of patients that may be candidates for livertransplantation include persons having one or more of the followingconditions, diseases, or disorders: acute liver failure, amyloidosis,bilirubin excretion disorders, biliary atresia, Budd-Chiari syndrome,chronic active autoimmune hepatitis, cirrhosis (either associated withviral hepatitis including hepatitis B and hepatitis C, alcoholiccirrhosis, or primary biliary cirrhosis), cholangitis, congenital factorVIII or IX disorder, copper metabolism disorders, cystic fibrosis,glycogenesis, hypercholesterolemia, lipidoses, mucopolysaccharidosis,primary sclerosing cholangitis, porphyrin metabolism disorders, purineand pyrimidine metabolism disorders, and primary benign and malignantneoplasms, especially of the liver and intrahepatic bile ducts, biliarysystem, biliary passages, or digestive system.

The clinical criteria for the identification of a patient or patientpopulation in need of, or likely to benefit from, a liver transplant canbe determined according to the knowledge and skill in the art. Suchcriteria may include, for example, one or more of the followingsymptoms: fatigue, weight loss, upper abdominal pain, purities,jaundice, liver enlargement, discolored urine, elevated alkalinephosphatase, and gamma glutamylpeptidase activity, elevated bilirubinlevels, decreased serum albumin, elevated liver-specific enzymes, lowbile production, increased blood urea nitrogen, increased creatinineand/or presence of anti-neutrophil cytoplasmic antibodies (ANCA) titers,recurrent variceal hemorrhage, intractable ascites, spontaneousbacterial peritonitis, refractory encephalopathy, severe jaundice,exacerbated synthetic dysfunction, sudden physiologic deterioration, andfulminant hepatic failure.

Kidney (Renal) Transplants

The compositions and methods of the invention are useful for treating orpreventing GVHD, and humoral or cellular rejection in a renal transplantrecipient. As used herein, the term “renal transplant” encompasses thetransplant of a kidney and the combined transplant of a kidney and apancreas. In particular embodiments, the rejection is characterized asacute rejection or chronic rejection.

In one embodiment, the compositions and methods of the inventioncomprise or are used in combination with a pre-transplant conditioningregimen. In one embodiment, a single dose of one or more of thecompositions of the present invention is effective in the patient orpatient population. In another embodiment, multiple doses of one or moreof the compositions of the invention are effective in the patient orpatient population. In one embodiment, a single dose of one or more ofthe compositions of the present invention is administered in combinationwith one or more immunosuppressive agents and is effective in thepatient or patient population.

In certain embodiments, the compositions and methods of the inventionare for treating or preventing GVHD and graft rejection in a patienthaving received a renal transplant. In one embodiment, the patient hasnot yet exhibited clinical signs of rejection. In a related embodiment,the compositions and methods of the invention comprise or are used incombination with a maintenance regimen for the prevention of graftrejection in the transplant recipient. In one embodiment, thecompositions and methods of the invention are for the treatment ofsubclinical humoral rejection. In a related embodiment, the patient orpatient population in need of treatment for a subclinical humoralrejection is indicated by the detection of Cd4 deposition or cellularinfiltration in a biopsy from the graft, or by the detection ofcirculating anti-HLA antibodies. In one embodiment, the compositions andmethods of the invention are for the treatment of subclinical cellularrejection.

In one embodiment, the compositions and methods of the inventioncomprise or are used in combination with a therapeutic regimen for thetreatment of an acute or chronic rejection episode in a transplantrecipient. In one embodiment, the patient or patient population in needof treatment for an acute or chronic rejection episode is identified bythe detection of one or more clinical indicators of rejection. Inspecific embodiments, the one or more clinical indicators of rejectionare detected one to six weeks post-transplantation. In one embodiment,the one or more clinical indicators of rejection are detected 6, 12, 18,24, 36, 48, or 60 months post-transplantation. In a preferredembodiment, the acute rejection is biopsy-confirmed acute humoral orcellular rejection.

In one embodiment, one or more of the compositions of the inventioncomprise a therapeutic regimen for the treatment of acute rejection. Ina particular embodiment, the therapeutic regimen further comprises oneor more of the following: plasmapheresis, tacrolimus/mycophenolate,intravenous immunoglobulin, and immunoadsorption with protein A. In oneembodiment, the patient has been on an immunosuppressive protocol priorto the development of the rejection. In a particular embodiment, theimmunosuppressive protocol includes one or more of cyclosporine,azathioprine, and steroid therapy.

Clinical indicators of acute humoral and cellular rejection are known inthe art and include, for example, a sudden severe deterioration of renalfunction, the development of oliguria, and compromised renal perfusion.Additional indicators include, for example, inflammatory cells inperitubular capillaries on biopsy and circulating donor-specificalloantibodies. In one embodiment, the patient presents with one or moreof the following diagnostic criteria for a humoral rejection of a renalallograft: (1) morphological evidence of acute tissue injury; (2)evidence of antibody action, such as C4d deposits or immunoglobulin andcomplement in arterial fibrinoid necrosis; and (3) detectablecirculating antibodies against donor HLA antigens or donor endothelialantigens. In one embodiment, the patient presents with all three of theabove diagnostic criteria.

In one embodiment, the patient presents with one or more of thediagnostic criteria for acute humoral or cellular rejection and thecompositions of the present invention are used in combination with oneor more of the following immunosuppressive agents to treat acuterejection: intravenous immunoglobulin, anti-thymocyte globulins,mycophenolate mofetil, or tacrolimus. In another embodiment, thecompositions of the invention are used in combination with one or moreimmunosuppressive agents and a procedure for the removal ofalloantibodies from the patient, such as plasmapheresis orimmunoadsorption.

In one embodiment, the compositions and methods of the inventioncomprise or are used in combination with a therapeutic regimen for thetreatment of chronic renal allograft rejection. In one embodiment, oneor more of the compositions of the invention are used alone or incombination with one or more immunosuppressive agents, including forexample, monoclonal antibodies, tacrolimus, sirolimus, and mizoribin. Ina preferred embodiment, one or more of B10 effector cell compositions ofthe invention are used in combination with tacrolimus, mycophenolate, orother appropriate therapeutics.

Clinical indicators of chronic rejection in the kidneys are known in theart and may include, for example, arterial intiamal fibrosis withintimal mononuclear cells (chronic allograft vasculopathy), duplicationof the glomerular basement membranes (chronic allograft glomerulopathy),lamination of the peritubular basement membrane, C4d deposition inperitubular capillaries, inflammatory cell infiltrates andimmunopathology, and detectable circulating donor HLA-reactiveantibodies. In a preferred embodiment, the compositions and methods ofthe invention comprise or are used in combination with a therapeuticregimen to treat chronic rejection before graft lesions develop.

In another embodiment, the patient or patient population in need oftreatment is identified as having one or more clinical indicators oftransplant glomerulopathy. In a related embodiment, the compositions ofthe invention comprise or are used in combination with a therapeuticregimen comprising one or more therapeutic agents. In a preferredembodiment, the therapeutic regimen is effective to stabilize renalfunction and inhibit graft rejection. In a particular embodiment, theone or more therapeutic agents include angiotensin converting enzyme(ACE) inhibitors and/or receptor antagonists, intra-venousimmunoglobulin, anti-thymocyte globulins, mycophenolate mofetil, ortacrolimus. Preferably, the B10 effector cells of the invention are usedin combination with mycophenolate mofetil and tacrolimus, with orwithout other therapeutic agents. Plasmapheresis and other treatmentsmay also be used as part of the therapeutic regimen.

A patient or patient population in need of, or likely to benefit from, arenal transplant is identified according to the knowledge and skill inthe art. Examples of patients that may be candidates for renaltransplantation include patients diagnosed with amyloidosis, diabetes(type I or type 11), glomerular disease (e.g., glomerulonephritis),gout, hemolytic uremic syndrome, HIV, hereditary kidney disease (e.g.,polycystic kidney disease, congenital obstructive uropathy, cystinosis,or prune bell syndrome), other kidney disease (e.g., acquiredobstructive nephropathy, acute tubular necrosis, acute interstitialnephritis), rheumatoid arthritis, systemic lupus erythematosus, orsickle cell anemia. Other candidates for renal transplant includepatients having insulin deficiency, high blood pressure, severe injuryor burns, major surgery, heart disease or heart attack, liver disease orliver failure, vascular disease (e.g., progressive systemic sclerosis,renal artery thrombosis, scleroderma), vesicoureteral reflux, andcertain cancers (e.g., incidental carcinoma, lymphoma, multiple myeloma,renal cell carcinoma, Wilms tumor). Other candidates for renaltransplant may include, for example, heroin users, persons who haverejected a previous kidney or pancreas graft, and persons undergoing atherapeutic regimen comprising antibiotics, cyclosporin, orchemotherapy. The clinical criteria for the identification of a patientor patient population in need of, or likely to benefit from, a kidneytransplant can be determined according to the knowledge and skill in theart. Such criteria may include, for example, one or more of thefollowing: urinary problems, bleeding, easy bruising, fatigue,confusion, nausea and vomiting, loss of appetite, pale skin (fromanemia), pain in the muscles, joints, flanks, and chest, bone pain orfractures, and itching.

Cardiac Transplants

The compositions and methods of the invention are useful for treating orpreventing GVHD, and humoral or cellular rejection in a cardiactransplant recipient. In particular embodiments, the rejection is acuteor a chronic rejection. In one embodiment, the compositions and methodsof the invention comprise or are used in combination with a pre- orpost-transplant conditioning regimen.

In certain embodiments, the compositions and methods of the inventioncomprise or are used in combination with a therapeutic regimen for thetreatment of acute humoral or cellular rejection in a cardiac transplantrecipient. In a particular embodiment, the therapeutic regimen furthercomprises one or more of the following: plasmapheresis, intravenousimmunoglobulin, and antibody therapy. The patient or patient populationin need of treatment for an acute humoral rejection is identified by thedetection of one or more of the clinical indications of acute humoralrejection. Examples of clinical indicators of acute humoral rejectionmay include one or more of the following: hemodynamic dysfunction,defined by shock, hypotension, decreased cardiac output, and a rise incapillary wedge or pulmonary artery pressure. In a particularembodiment, the acute humoral rejection is diagnosed within 6, 12, 18,24, 36, 48, or 60 months post-transplantation.

In one embodiment, the compositions and methods of the inventioncomprise or are used in combination with a therapeutic regimen for theprevention of humoral or cellular rejection in a cardiac transplantrecipient. In one embodiment, the transplant recipient in need ofprophylaxis against rejection is identified as a patient or patientpopulation having one or more of the following risk factors: female sex,cytomegalovirus seropositivity, elevated response to panel reactiveantibodies, positive pre- and/or post-transplant crossmatch, andpre-sensitization with immunosuppressive agents.

In one embodiment, the compositions and methods of the invention are forthe treatment or prevention of graft deterioration in a heart transplantrecipient. In one embodiment, the transplant recipient in need oftreatment for, or prophylaxis against, graft deterioration is identifiedas a patient or patient population having one or more of the followingclinical indications of humoral or cellular rejection: deposition ofimmunoglobulin, C1q, C3, and/or C4d in capillaries, evidence ofCD68-positive cells within capillaries, and evidence of infiltration ofthe graft by inflammatory cells upon biopsy. In one embodiment, thecompositions of the present invention are used in combination with oneor more of the following immunosuppressive agents to treat graftdeterioration in a heart transplant recipient: intravenousimmunoglobulin, anti-thymocyie globulins, monoclonal antibodies,mycophenolate mofetil, or tacrolimus. In another embodiment, the B10effector cell compositions of the invention are used in combination withone or more immunosuppressive agents and a procedure for the removal ofalloantibodies from the patient, such as plasmapheresis orimmunoadsorption.

In one embodiment, the compositions and methods of the inventioncomprise or are used in combination with a therapeutic regimen for thetreatment of chronic cardiac rejection, preferably chronic allograftvasculopathy, also referred to as transplant coronary artery disease. Inanother embodiment, the compositions and methods of the inventioncomprise or are used in combination with a therapeutic regimen for theprevention of transplant coronary artery disease in a patient or patientpopulation at risk. The criteria for identifying a patient or patientpopulation at risk of developing transplant coronary artery disease areknown in the art and may include, for example, patients having poorlymatched transplants, patients who develop circulating anti-HLAantibodies, and patients who develop one or more clinical indications ofhumoral or cellular rejection early after cardiac transplant.

A patient or patient population in need of, or likely to benefit from aheart transplant is identified according to the knowledge and skill inthe art. Examples of patients that may be candidates for hearttransplantation include those who have been diagnosed with any of thefollowing diseases and disorders: coronary artery disease,cardiomyopathy (noninflammatory disease of the heart), heart valvedisease with congestive heart failure, life-threatening abnormal heartrhythms that do not respond to other therapy, idiopathic cardiomyopathy,ischemic cardiomyopathy, dilated cardiomyopathy, ischemiccardiomyopathy, and congenital heart disease for which no conventionaltherapy exists or for which conventional therapy has failed.

The clinical criteria for the identification of a patient or patientpopulation in need of, or likely to benefit from, a heart transplant canbe determined according to the knowledge and skill in the art. Suchcriteria may include, for example, one or more of the following:ejection fraction less than 25%, intractable angina or malignant cardiacarrhythmias unresponsive to conventional therapy, and pulmonary vascularresistance of less than 2 Wood units. In addition, the patient orpatient population in need of a heart transplant may be identified byperforming a series of tests according to the knowledge and skill in theart. Such tests include, for example, resting and stressechocardiograms, EKG, assay of blood creatinine levels, coronaryarteriography, and cardiopulmonary evaluation including right- andleft-heart catheterization.

Lung Transplant

The compositions and methods of the invention are useful for treating orpreventing GVHD, and humoral or cellular rejection in a lung transplantrecipient. In particular embodiments, the refection is characterized asan acute or a chronic rejection. In one embodiment, the compositions andmethods of the invention comprise or are used in combination with apre-transplant conditioning regimen. A patient or patient population inneed of, or likely to benefit from, a lung transplant is identifiedaccording to the knowledge and skill in the art. Examples of patientsthat may be candidates for lung transplantation include patients havingone of the following diseases or conditions: bronchiectasis, chronicobstructive pulmonary disease, cystic fibrosis, Eisenmenger syndrome orcongenital heart disease with Eisenmenger syndrome. emphysema,eosinophilic granuloma of the lung, or histiocytosis X, inhalatiodbumtrauma, lymphangioleiomyomatosis (LAM), primary pulmonary hypertension,pulmonary fibrosis (scarring of the lung), or sarcoidosis.

The clinical criteria for the identification of a patient or patientpopulation in need of, or likely to benefit from, a lung transplant canbe determined according to the knowledge and skill in the art. Suchcriteria may include, for example, one or more of the following: chronicobstructive pulmonary disease (COPD) and alpha1-antitrypsin deficiencyemphysema characterized by one or more of the following indicators:postbronchodilator FEV1 of less than 25% predicted, resting hypoxemia,i.e., Pa0₂ of less than 55-60 mm Hg, hypercapnia. secondary pulmonaryhypertension, a rapid rate of decline in FEV1, or life-threateningexacerbations; cystic fibrosis characterized by one or more of thefollowing indicators: postbronchodilator FEV1 of less than 30%predicted, resting hypoxemia, hypercapnia, or increasing frequency andseverity of exacerbations; idiopathic pulmonary fibrosis characterizedby one or more of the following indicators: vital capacity (VC) and TLCof less than 60-65% predicted, and resting hypoxemia; secondarypulmonary hypertension characterized by clinical, radiographic, orphysiologic progression while on medical therapy; primary pulmonaryhypertension characterized by one or more of the following indicators:NYHA functional class III or IV, mean right atrial pressure of greaterthan 10 mm Hg, mean pulmonary arterial pressure of greater than 50 mmHg, cardiac index of less than 2.5 L/min/m², and failure of therapy withlong-term prostacyclin infusion.

Treatment of Subjects Receiving Biologics

Methods of treating subjects receiving recombinant, therapeutic orxenogeneic protein(s) are also provided. The methods includeadministering a therapeutically effective amount of the B10 cellsdescribed herein to a subject in need of treatment for a genetic,transplantation, allergy, inflammation, or autoimmune disorder. Inparticular the B10 cells may be co-administered with a biologic or othertherapeutic to which a subject may develop or has developed anti-drugantibodies.

As the number of biologic therapies that reach the clinics continues toincrease, there is an increasing appreciation that recipients frequentlydevelop immune responses to these drugs, which has a potential clinicalimpact on drug efficacy. Immune responses to biologics are generallymonitored by detection and characterization of anti-drug antibodies(ADA) and assessing ADA associations with drug exposure, efficacy andsafety. The detection of ADA does not necessarily mean that there willbe clinical consequences. There are, however, an increasing number ofexamples where ADA can challenge drug efficacy and patient safety.

Thus the B10 cell compositions may be used in combination with(co-administration, provided before or after) a biologic therapy toavoid production of ADA.

Assessing B10 Cell Function

Methods of assessing the B10 cell function in a subject are alsoprovided herein. The methods include harvesting B cells from a subjectas discussed above for the methods of expanding B cells ex vivo andculturing the cells in the presence of a CD40 agonist and IL-21. Any ofthe methods for expanding B cells described herein may be utilized toassess B10 cell function in the subject. After culturing with IL-21, thecells are then assayed to determine if the cells are capable ofproducing IL-10. The percentage of cells in the culture capable ofmaking IL-10 and/or the amount of IL-10 produced by the cells may bedetermined. The determination may be made either after culturing withIL-21 or after culture with another stimulatory molecule or combinationof molecules such as an antigen or LPS. The determination of thepercentage of cells producing IL-10 or the amount of IL-10 beingproduced may be determined by any method available to one of skill inthe art.

The amount of IL-10 or percentage of cells producing IL-10 may bedetermined and compared to a control. Suitably the control is a normalcontrol comprising B cells from a healthy donor treated similarly to thecells obtained from the subject. Alternatively the control may berepresented by a numeric range over which healthy donor cells areexpected to fall. The B10 cell function of the subject may be normal,overactive or deficient as compared to a healthy donor. If B10 cellfunction is not normal, the method may be used to diagnose the subjector indicate that the subject either has or is likely to have a diseaseor condition affecting B10 cell function.

The following examples are meant only to be illustrative and are notmeant as limitations on the scope of the invention or of the appendedclaims. All references cited herein are hereby incorporated by referencein their entireties.

EXAMPLES

To identify signals that regulate B10 cells in vivo, purified B cellswere cultured with cytokines known to influence B cell function.Stimulation with IL-21, but not IL-4, -6, -10, -12, -23 or -27, induced2.7- to 3.2-fold higher B10 cell frequencies and 4.4- to 5.3-fold moreIL-10 secretion (p<0.01) at 48 and 72 h, respectively, whileinterferon-γ (IFN-γ) or transforming growth factor-β (TGF-β) reducedIL-10⁺ B cell frequencies by 56% (p<0.05; FIG. 1A). In fact, IL-21induced B10 cells to produce IL-10 without a need for in vitrostimulation (FIG. 5A) and induced B cell IL-10 secretion at levelssimilar to lipopolysaccharide (LPS) stimulation (FIG. 1A). IL-21 alsoinduced a 3-fold increase in IL-10⁺ B cells within the spleenCD1d^(hi)CD5⁺ B cell subset that is enriched for B10pro and B10 cells,but it did not induce significant numbers of IL-10⁺ B cells among theCD1d^(lo)CD5⁻ subset (FIG. 1B). There was little if any detectable Bcell division in the 48 or 72 h assays when the cells were cultured withcytokines alone. Even when mitogens such as anti-IgM antibody or LPSwere added to induce B cell proliferation, there was still littleproliferation at 48 h, although B10 cells are the most proliferativecells at 72 h. IL-21 did not promote B10 cell survival, but insteadaccelerated the apoptosis of non-B10 cells while B10 cells werepredominantly spared. The net result was that B10 cell numbers werepreferentially increased by IL-21 relative to other cytokines.Consistent with this, IL-21 induces either B cell apoptosis ordifferentiation in a context-dependent manner, driving the in vitrodifferentiation and expansion of more completely activated B cells. SeeSpolski, R. & Leonard, W. J. Interleukin-21: basic biology andimplications for cancer and autoimmunity. Annu Rev Immunol 26, 57-79(2008) and Ozaki, K. et al. Regulation of B cell differentiation andplasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6.J. Immunol. 173, 5361-5371 (2004). IL-21 is also known to be a potentinducer of T cell IL-10 production, and T cell-derived IL-21 playsmultiple important roles in B cell effector function. Both B10 andnon-B10 cells expressed cell surface IL-21 receptor (IL-21R) at similarlevels (FIG. 1C). Despite this, ex vivo B10 and B10+B10pro cell andCD1d^(hi)CD5⁺ B cell numbers were similar in IL-21R-deficient(IL-21R^(−/−)), wild type, MHC-II^(−/−) and CD40^(−/−) mice (FIG. 5B-D).However, IL-21R expression was required for B10 cell expansion followingmyelin oligodendrocyte glycoprotein peptide (MOG₃₅₋₅₅) immunizations toinduce EAE (FIG. 1D). Thus, IL-21R-generated signals induced B cellIL-10 secretion in vivo and in vitro and were required for B10 cellexpansion in vivo.

Whether B10 cells require IL-21 to induce their regulatory function invivo was determined by the adoptive transfer of IL-21R^(−/−) B cellsinto CD19^(−/−) mice before the induction of EAE by MOG₃₅₋₅₅immunization. Because CD19^(−/−) mice are B10 cell-deficient (FIG. 1d ),their EAE disease severity is worse (FIG. 2A). The adoptive transfer ofwild type CD1d^(hi)CD5⁺ B cells normalized EAE severity in CD19^(−/−)mice. By contrast, the transfer of CD1d^(hi)CD5⁺ B cells fromIL-10^(−/−) or IL-21R^(−/−) mice or wild type CD1d^(lo)CD5⁻ non-B10cells did not alter disease. Because CD4⁺ T cells are a major source ofIL-21, we determined whether cognate B10-T cell interactions alsocontrolled B10 cell-mediated suppression of EAE. The transfer ofCD1d^(hi)CD5⁺ B cells from MHC-II^(−/−) or CD40^(−/−) mice intoCD19^(−/−) mice before MOG immunizations did not reduce EAE diseaseseverity (FIG. 2A, bottom right two panels). CD1d^(lo)CD5⁻ B cells fromIL-21R^(−/−), CD40^(−/−) or MHC-II^(−/−) mice were also without effect.EAE is also exacerbated in wild type mice depleted of mature B cells byCD20 mAb. However, transfer of CD1d^(hi)CD5⁺ B cells from CD20^(−/−)mice but not MHC-II^(−/−) CD20^(−/−) mice normalized disease severity inthis model, and CD1d^(lo)CD5⁻ B cells from CD20^(−/−) or MHC-II^(−/−)CD20^(−/−) mice were without effect (FIG. 2B). Similarly, the adoptivetransfer of in vitro activated CD1d^(hi)CD5⁺ B cells from wild type micesignificantly reduced EAE disease severity in wild type mice, whereasactivated MHC-II^(−/−) CD1d^(hi)CD5⁺ or wild type CD1d^(lo)CD5⁻ B cellshad no effect (FIG. 2C). Thus, regulatory B10 cell function requiredIL-10 expression, IL-21R signaling, as well as CD40 and MHC-IIinteractions, thereby potentially explaining antigen-specific B10 celleffector function.

IL-10 produced by activated CD1d^(hi)CD5⁺ B cells inhibitsantigen-specific CD4⁺ T cell IFN-γ and TNF-α expression in vitro. Todetermine whether cognate B10-T cell interactions regulateantigen-specific T cell proliferation in vivo, B10 cell function wasassessed in MOG₃₅₋₅₅-immunized CD19^(−/−) mice following the adoptivetransfer of dye-labeled CD4⁺ T cells from transgenic mice expressingantigen receptors (TCR^(MOG)) specific for MOG₃₅₋₅₅ peptide. SeeBettelli, E. et al. Myelin oligodendrocyte glycoprotein-specific T cellreceptor transgenic mice develop spontaneous autoimmune optic neuritis.J. Exp. Med. 197, 1073-1081 (2003). CD1d^(hi)CD5⁺ B cells from naïvewild type mice significantly reduced TCR^(MOG) CD4⁺ T cell proliferationas measured by in vivo dye dilution (FIG. 3A). CD1d^(hi)CD5⁺ B cellsobtained from mice with EAE were even more potent inhibitors of T cellproliferation, while CD1d^(lo)CD5⁻ B cells from wild type mice orCD1d^(hi)CD5⁺ B cells from IL-10^(−/−), CD40^(−/−), or MHC-II^(−/−) micewere without effect. CD1d^(hi)CD5⁺ B cells from naïve orantigen-experienced wild type mice also significantly reduced TCR^(MOG)CD4⁺ T cell IFN-γ and IL-17 production in MOG₃₅₋₅₅-immunized CD19^(−/−)mice, while CD1d^(hi)CD5⁺ B cells from IL-10^(−/−), CD40^(−/−) orMHC-II^(−/−)mice did not (FIG. 3B). The ability of B10 cells to inhibitT cell IL-17 production is particularly important since pathogenicT_(H)17 T cells induce EAE and can produce IL-21. The majority of Tfollicular helper cells isolated from mice with MOG₃₅₋₅₅-induced EAEalso express IL-21, and CD19^(−/−) mice have T follicular helper cells(FIG. 6). Thus, B10 and T cells may require intimate interactions duringreciprocal IL-10 and IL-21 production to optimally regulateantigen-specific disease (FIG. 3C).

Although T follicular helper cells are a likely source of IL-21, thereare currently no indications that B10 cells are germinal centerconstituents and most data argue against this. First, B10 cell BCRs arepredominantly germline and contain modest frequencies of IgV_(H) andIgV_(L) mutations. Maseda, D. et al. Regulatory B10 cells differentiateinto antibody-secreting cells after transient IL-10 production in vivo.J. Immunol. 188, 1036-1048 (2012). Second, B10 cell numbers expand earlyduring the induction of EAE, prior to the generation of germinalcenters. Matsushita, T., Horikawa, M., Iwata, Y. & Tedder, T. F.Regulatory B cells (B10 cells) and regulatory T cells have independentroles in controlling EAE initiation and late-phase immunopathogenesis.J. Immunol. 185, 2240-2252 (2010). Third, B10 cell GL-7 expressionresembles spleen follicular B cells and not GL-7^(high) germinal centerB cells. Matsushita, 2010. Furthermore, a hallmark of transgenic micewith dramatically expanded B10pro+B10 cell numbers is the absence ofgerminal centers and little if any B cell isotype switching, even afterimmunizations. Poe, J. C. et al. Amplified B lymphocyte CD40 signalingdrives regulatory B10 cell expansion in mice. PLoS ONE 6, e22464 (2011).B10 cells in these mice are also located throughout both the marginalzone and follicular areas of the spleen.

To verify that T cell-derived IL-21 and CD40 signals drive B10 cellexpansion and IL-10 production, B cells were cultured using conditionsto promote mouse B10 cell expansion in vivo and B cell expansion invitro. See Poe, 2011 and Nojima, T. et al. In-vitro derived germinalcentre B cells differentially generate memory B or plasma cells in vivo.Nature Comm. 2, 465 (2011). B cells were cultured on monolayers ofNIH-3T3 cells expressing the T cell ligand for CD40 (CD154) and BLyS inthe presence of IL-4 for 4 days to induce B10pro cell maturation intoIL-10-competent B10 cells. The B cells were then cultured on freshNIH-3T3-CD154/BLyS cells with exogenous IL-21 for 5 days, which were allessential to optimally expand B10 cells and induce IL-10 production(FIG. 4A). After the 9 day culture period, B cell and B10 cell numberswere increased by 25,000- and 4,000,000-fold, respectively, with 38% ofthe B cells actively producing IL-10 (FIG. 4B). The vast majority ofIL-10⁺ B cells in the cultures expressed CD5 (FIG. 4C), facilitatingtheir purification and underscoring the dramatic effect of IL-21 on B10cell numbers in vitro.

In vitro-expanded CD5⁺ B10 cells retained their regulatory function. Thetransfer of CD5⁺ B10 cells dramatically reduced EAE disease severity inwild type mice, even when given after the appearance of diseasesymptoms, while CD5⁻ B cells were without effect (FIG. 4D). Although thein vitro expansion of B10 cells required both IL-21R and CD40 signals,MHC-II expression was not required (FIG. 4E). However, in vitro-expandedMHC-II^(−/−) CD5⁺ B10 cells and IL-10^(−/−) CD5⁺ B cells did notregulate EAE disease severity (FIG. 4F), further documenting arequirement for IL-10 and cognate interactions in the regulation of Tcell-mediated disease. B10 cells did not expand during in vitro culturesof B cells from CD19^(−/−) mice or MD4 transgenic mice that have a fixedBCR specific for egg lysozyme (FIG. 4E), further underscoring theimportance of BCR specificity and signaling in B10 cell generation.Otherwise, in vitro-expanded B10 effector cells were potent regulatorsof both disease initiation and progression.

This study demonstrates that CD40 signals induce B10pro cell acquisitionof IL-10 competence with IL-21 driving B10 cell expansion and effectorcell generation. These critical checkpoints in B10 cell development maylead to localized IL-10 production that blunts antigen-specific T cellresponses during cognate interactions (FIG. 3C) without untowardimmunosuppression. Transient IL-10 production by B10 cells in vivo mayfurther restrict the effects of IL-10 secretion. B10 effector cells mayalso regulate T cell responses to autoantigens in addition to MOG onceinflammation and tissue destruction are initiated by MOG₃₅₋₅₅immunization. Since human and mouse B10 cells are also potent regulatorsof macrophage and dendritic cell function, T cell induction of B10effector cells may also contribute to EAE resolution by restrainingmonocyte and dendritic cell activation. These collective results mayexplain in part why EAE is exacerbated in the absence of IL-21signaling. By contrast, TGF-β and IFN-γ may counterbalance B10 cellexpansion in vivo based on the current in vitro findings (FIG. 1A).Regulatory T cells provide an independent layer of regulation during EAEsince their expansion, accumulation in the central nervous system, andsuppressive activity are normal when B10 cells are absent. The in vitrorecapitulation of these collective signals induced a severalmillion-fold expansion of B10 cells and their functional maturation intopotent B10 effector cells that reversed established autoimmune disease(FIG. 4). In addition to BCR specificity, MHC-II expression remained animportant checkpoint for B10 effector cell regulatory function duringEAE (FIG. 4F), as first described for regulatory type II monocytes.Since autoimmunity has multigenic origins and autoantigens vary betweenpatients and disease, in vitro expansion of the rare pool of human bloodB10pro and B10 effector cells may provide a potent future immunotherapyfor individuals with severe autoimmune disease.

Human B10 Cell Expansion In Vitro.

To determine whether IL-21 and CD40 signals drive human B10 cellexpansion in vitro, purified blood B cells (1×10⁶/ml) were cultured onconfluent monolayers of mitomycin C-treated NIH-3T3 cells expressing themouse T cell ligand for CD40 (mCD154) and human BLyS (hBLyS) in thepresence of human IL-4 (2 ng/ml) for 7 days to induce B10pro cellmaturation into IL-10-competent B10 cells. The B cells were thencultured on fresh NIH-3T3-mCD154/hBLyS cell monolayers with exogenoushuman IL-21 (10 ng/ml) for 5 days. NIH-3T3 cells expressing mouse CD154were used for both the mouse and human studies because mouse CD154 bindshuman CD40 (Bossen et al. 2006 J. Biol. Chem. 281:13964-13971) and mouseCD154 can induce signals through both mouse and human CD40 (Armitage etal. 1992 Nature 357:80-82 and Yasui et al., 2002 Intl. Immunol.14:319-329). By contrast, human CD154 does not bind mouse CD40 (Bossenet al., 2006). Human BLyS was used for both mouse and human B cellexpansion because human BLyS appears to bind both human and mousereceptors (BCMA, TACI and BAFF-R) similarly (Bossen et al. 2006)). Afterthe 12 day culture period, B cell numbers increased by 130 (±17)-fold,while B10 cell numbers were increased by 5-6,000-fold, with 13-16% ofthe B cells expressing IL-10 following 5 h of stimulation with phorbolester, ionomycin and Brefeldin-A (FIG. 7). These results parallel ourearly results with in vitro expansion of mouse B cells and B10 effectorcells, and indicate that this culture system is translatable to humans.

As occurred with our mouse studies, additional refinement of the culturesystem and protocols will be required for maximal human blood B cell andB10 effector cell expansion. These findings are not unanticipated ashuman B cell subpopulations respond differently to graded levels ofCD40-CD154 interactions (Neron et al. 2005 Immunology 116:454-463) andvarious forms of CD40 ligand used for in vitro stimulation of human Bcells, such as membrane-associated CD154, soluble trimeric, dimeric ormonomeric CD154 proteins or anti-CD40 antibodies, produce distinctfunctional responses in a wide range of B cell activities (Fanslow etal. 1994 Sem. Immunol. 6:267-278). Furthermore, human CD40 costimulationis differentially affected by cytokines. For example, CD40 ligation inthe presence of IL-4 co-stimulates human B cells to secrete IgE andIgG4, while CD40 ligation in the presence of IL-2 or IL-10 induces humanblood or tonsil B cells to secrete other Ig isotypes (IgM, IgG1, IgG2,IgG3 and IgA).

Feeder Cell Supplemental Factors Involved in Ex Vivo B Cell Expansion.

Our findings during in vitro expansion of mouse and human B cells andB10 effector cells demonstrate that this culture system is translatableto humans, but is not solely dependent on the addition of exogenous IL-4and IL-21, or feeder cell expression of CD154 and BLyS. Each of thesefactors must be optimized for maximal B cell and B10 cell expansion anddifferent B cell subpopulations respond differently to graded levels ofCD4O-CD154 interactions (Neron et al., 2005 Immunology 116:454-463).Furthermore, it is anticipated that additional factors can be added tothe cultures or expressed by the feeder cells to further optimize B celland B10 cell expansion.

Most important is that not all mouse NIH-3T3 (Swiss) fibroblasts, mouse3T3-Balb/c fibroblasts, or human EA Hy.926 endothelial cells (Li et al.,1998 J. Exp. Med. 188:1385-1390 and Edgell et al., 1983 Proc. Natl.Acad. Sci., USA 80:3734-3737) are equivalent. For example, CD154⁺ BLyS⁺NIH-3T3 and 3T3-Balb/c fibroblasts were able to induce dramatic B cellactivation and proliferation, with the majority of activated B cellsadhering to the stromal cells and forming large grape-like clusters. Bycontrast, EA Hy.926 endothelial cells were only able to induce dramaticB cell activation and the activated B cells did not adhere to thestromal cells. However, we subsequently determined that EA Hy.926 cellsdid not express vascular cell adhesion molecule 1 (VCAM-1), while bothfibroblast cell lines constitutively expressed VCAM-1 (Table 1; below).Importantly, we have determined that B cell adhesion to stromal cells isrequired for their initial activation and expansion in the culturesystem. VCAM-1 is critical for molecular interactions between stromalcells and B cell precursors that lead to B lymphohematopoiesis (Kincadeet al., 1989 Annu. Rev. Immunol. 7:111-143 and Kincade 1992 SeminImmunol 3:379-390). Similarly, CD44 binding of hyaluronic acid andpotentially other molecules is required for B cell adherence to bonemarrow stromal cells and subsequent lymphohematopoiesis in long termbone marrow cultures (Lesley et al. 1992 J. Exp. Med. 175:257-266.).Both NIH-3T3 and 3T3-Balb/c fibroblasts constitutively express CD44(Table 1). Thus, effective stromal cells must express appropriate cellsurface molecules and/or provide an appropriate substrate for B cellattachment. Thereby, stromal cells for optimal B cell and B10 cellexpansion would minimally express CD154, BLyS, VCAM-1 and CD44, or otherfunctionally equivalent molecules, with exogenous IL-4 and IL-21 addedor these cytokines produced by the stromal cells at optimized levels.

Transfected stromal cell cultures that were functionally selected foroptimal CD154 and BLyS expression were able to support B cell expansion,but there was tremendous heterogeneity between different batchtransfectants, individual clones, and their subclones despite similarCD154 and BLyS expression. The tremendous heterogeneity in abilities ofdifferent transfectants to support B cell expansion was eventuallyexplained by our finding that there was extreme cellular and functionalheterogeneity within each CD154⁺ BLyS⁺ stromal cell population. Althoughunexpected, it is well recognized that stromal cells can expresssignaling molecules and secrete cytokines, can respond to some of thosesignaling molecules and cytokines themselves, can respond to exogenouscytokines added to the cultures, and that they can retaindifferentiation potential depending on their culture conditions.

Within our transfected stromal cell populations, we determined that onlya subset of the cells were able to support robust B cell adhesion andclonal expansion, with a frequency that was commonly <1% of stromalcells despite all of the cells expressing CD154 and secreting BLyS.These stromal cell cultures were generally able to induce B cellexpansion by <20-fold. However, mechanical isolation allowed us toisolate optimal cells that uniformly supported B cell expansion over abroad range up to 25,000-fold as described above (FIG. 4). Stromal cellsoptimized for maximal B cell and B10 cell growth can be mechanicallyisolated based on phenotypic, morphologic and growth characteristics.For example, it was possible to enrich for stromal cells that had agreater capacity to support B cell expansion by isolating individualstromal cells that supported B cell adhesion and rosette formation.Additional mechanical means for isolating optimal stromal cells for Bcell expansion can include single cell-cloning techniques, flowcytometry isolation of cells based on their expression or loss of cellsurface molecules, and/or other techniques known to those with skill inthe art, with the subsequent functional identification of stromal cellsthat support optimal B cell expansion.

The bone marrow microenvironment and stromal cells can either positivelyor negatively influence whether a given B cell precursor or B cellproliferates, differentiates, or undergoes apoptosis. For example,several stromal cell lines that support lymphocyte outgrowth suppressthe spontaneous apoptosis of pre-B cells by as much as 90%, while otherstromal cell clones can induce lymphocyte apoptosis, or can appearinert. Borghesi et al. 1997 J Immunol 159:4171-4179. Similarobservations have been made for the stromal cells used for B cellexpansion in our studies. As examples, three representative clonesexpressing mouse CD154 and human BLyS are shown with one transfectedclone from 3T3-Balb/c parental cells and two transfected clones (1 and2) derived from parental NIH-3T3 cells (Table 1). At the time of thesestudies, clone #1 3T3-Balb/c cells supported optimal B cell expansion incomparison with clones 1 and 2 of NIH-3T3 cell origin, which wererelatively less effective. Microarray analysis of these three clones incomparison with their parental 3T3 cells demonstrated considerablemolecular heterogeneity between the cells cultured under identicalconditions. The expression of some molecules was either upregulated ordown-regulated, which correlated with optimal B cell expansion. However,the level of molecular heterogeneity between sub-clones was the mostunexpected finding. Examples of the molecular differences that arelikely to be functional are illustrated below.

Molecules that were upregulated in 3T3 cells with the potential tosignificantly expand B cells included CD24, also known as heat-stableantigen in the mouse. CD24 is a glycosylphosphatidylinositol-anchoredmembrane protein of heterogeneous molecular weight ranging from 30 to 70kDa. The mature protein is only 27 to 30 amino acids long, and most ofthe molecular weight of the protein consists of extensive N- andO-linked glycosylation. CD24 is expressed by B cells and theirprecursors and neutrophils, in neuronal tissue, and in certainepithelial cells. CD24 functions as a mucin-like adhesion molecule thatcan facilitate and regulate cell-cell interactions. Increased stromalcell IL-7 expression by clone #1 3T3-Balb/c cells may be critical to B10and B cell expansion. IL-7 is normally made at extremely small levels bystromal cells, but it is an essential stimulus for early B cellprecursor replication, and other critical developmental functions. Clone#1 3T3-Balb/c cells also expressed Macrophage stimulating 1 (Mst1), alsoknown as Ste20-like kinase or STK4 (serine threonine kinase 4), thehuman ortholog of Drosophila Hippo. STK4 is a central caspase3-activated constituent of a highly conserved pathway controlling cellgrowth and apoptosis. Importantly, lymphocytes and neutrophils fromSTK4-deficient mice exhibit enhanced loss of mitochondrial membranepotential and increased susceptibility to apoptosis. Mst1 also hascrucial roles in lymphocyte adhesion to endothelial cells duringlymphocyte trafficking in vivo and Mst1^(−/−) mice have hypotrophicperipheral lymphoid tissues and reduced marginal zone B cells in thespleen. Thymic stromal lymphopoietin (Tslp) protein is primarilyproduced by certain stromal cells and fibroblasts and acts on myeloidlineage cells to produce factors that influence T cell lineagedevelopment and certain T cell subsets including regulatory T cells.TSLP may also support B cell differentiation from fetal hematopoieticprogenitors. TSLP is proposed to signal through a heterodimeric receptorcomplex composed of the TSLP receptor and the IL-7Ra chain, suggestingthat TSLP and IL-7 may influence some signaling pathways in common.

Multiple stromal cell molecules are likely to negatively influence Bcell proliferation, differentiation, and interactions with stromalcells, or induce their apoptosis. CD99 is expressed by endothelial cellsas well as most leukocytes, including B cells. CD99 functions as anadhesion molecule, and also interacts with the intracellular moleculecyclophylin A that is intricately involved in inflammatory signalingpathways. Homotypic CD99-CD99 interactions have been shown betweenmonocytes and endothelial cells during diapedesis. CD99 signaling may bedetrimental to B cell expansion as CD99 ligation on early B cells usingan anti-CD99 monoclonal antibody induces apoptosis. Likewise, CD99signaling induces the apoptosis of developing T cells in the thymus.Interactions between the mouse homologue of CD99 (designated D4) and itsligand, paired immunoglobulin-like type 2 receptor (PILR) widelyexpressed by leukocytes, is a major mechanism inducing thymocyteapoptosis.

As a second example, transforming growth factor beta-induced (Tgfbi)expression was down-regulated by both clone #1 3T3-Balb/c cells andclone #1 NIH-3T3 cells in comparison with their parental cells.Transforming growth factor beta-induced (TGFBI) protein is a secretedRGD-containing protein induced by transforming growth factor-beta thatbinds to type I, II and IV collagens and may thereby inhibit celladhesion. RGD motifs are found in many extracellular matrix proteinsthat modulate cell adhesion and the motif serves as a ligand recognitionsequence for several integrins during cell-collagen interactions. TGFBIcan inhibit adhesive interactions regulating the invasive growth ofmelanoma cells. The loss of TGFBI expression has been implicated in cellproliferation, tumor progression, and angiogenesis.

In a third example, stromal cell CXCR7 transcripts were down-regulatedin both clone #1 3T3-Balb/c cells and clone #1 NIH-3T3 cells. CXCR7(formerly RDC1) functions as a receptor for the CXCL12 (formerly SDF-1)chemokine that binds B cells and can regulate a spectrum of normal andpathological processes. CXCR7 can function as a scavenger receptor forCXCL12 that is also normally produced by both parental 3T3 cell lines.Thereby, stromal cell loss of CXCR7 expression may facilitate CXCL12binding to B cells in the culture system.

Cell surface differentiation-regulating proteins may also becounterproductive to optimal B cell expansion. Delta-like 1 and Jagged 1expression and their shared receptor Notch 1 were down-regulated onclone #1 3T3-Balb/c cells in comparison with untransfected parentalcells. Notch, Delta-like and Jagged proteins play pivotal roles in Bcell development and activation. For example, Notch-1 engagement on Bcells by Delta-like 1 expressed on stromal cells promotes B celldifferentiation into antibody-secreting cells, while Notch-1interactions with Jagged-1 are inhibitory in this process. Also,Notch-Delta-like 1 interactions act in synergy with B cell antigenreceptor and CD40 signaling to enhance B cell proliferation and isotypeswitching. In addition, marginal zone B cell development criticallyrequires Delta-like 1 interactions with B cell-expressed Notch-2. Otheraspects of B cell development are negatively regulated byNotch/Delta-like/Jagged interactions. For example, 3T3 fibroblastsexpressing Delta-like 1 have been reported to act as stromal cells foradipocyte differentiation, but only support early B cell differentiationwhen Delta-like expression is suppressed or if interleukin-7 (IL-7) isalso provided to the cultures. Therefore, the role of theNotch/Delta-like/Jagged axis in the development and function of B10cells may be complex, but these proteins may be critical regulators of Bcell and B10 cell expansion.

Stromal cells for maximal B cell and B10 cell expansion may therebyoptimally express appropriate densities of CD154, BLyS, VCAM-1 and CD44,or other functionally equivalent molecules, along with optimalexpression of some or all of the molecules outlined in Table 1 incombination with exogenous IL-4 and IL-21 added to the cultures. As eachof the molecules that influence B cell and B10 cell expansion are betterdefined, it will be critical to evaluate the function of each moleculein combination with other factors endogenously expressed by the stromalcells and exogenous stimuli added to the cultures. The cumulativeeffects of these positive and negative regulators will also depend onthe timing in which these signals are presented during B cell and B10cell expansion. Thereby, a functional definition of optimal stromalcells for B cell and B10 cell expansion is currently more operable thana molecular definition that includes all factors and pathways that arerequired to maximal B cell and B10 cell expansion.

TABLE 1 Transcripts expressed by stromal cells optimized for B cell andB10 cell expansion. NIH 3T3-Swiss Gene Fold- 3T3-Balb/c clone clone Par-Symbol Gene Title Change Clone 1 Parent 1 2 ent Relative ++++ − ++ + −capacity for B10 cell expansion Upreg- ulated Cd24a CD24a antigen 10.133197 439 647 238 147 Cd24a CD24a antigen 6.05 5318 1317 1814 676 370Cd24a CD24a antigen 4.12 960 336 439 181 111 I17 interleukin 7 20.19 64783 24 24 23 I17 interleukin 7 3.66 67 17 24 17 17 Mst1 macrophage 4.90208 48 46 37 39 stimulating 1 Ts1p thymic stromal 1.23 82 20 355 74 38lymphopoietin Downreg: Cd99 CD99 antigen −9.26 67 682 902 545 448 Cd99CD99 antigen −9.43 65 631 892 529 481 Cd99 CD99 antigen −22.14 35 8991244 670 508 Cxcr7 chemokine −73.82 12 1684 55 2445 2543 (C-X-C motif)receptor 7 Dlk1 delta-like 1 −4.31 56 750 201 227 96 homolog(Drosophila) Dlk1 delta-like 1 −89.03 59 11502 8915 6262 1191 homolog(Drosophila) Jag1 jagged 1 −2.37 43 98 358 57 54 Jag1 jagged 1 −11.34 29493 1755 103 128 Jag1 jagged 1 −53.26 18 2094 5614 149 434 Notch1 Notchgene −12.74 50 599 754 661 547 homolog 1 (Drosophila) Notch1 Notch gene−4.01 68 242 318 265 272 homolog 1 (Drosophila) Tgfbi transforming−14.43 13 192 19 586 570 growth factor, beta induced Tgfbi transforming−14.62 16 182 26 785 772 growth factor, beta induced Tgfbi transforming−36.28 11 539 11 2062 2196 growth factor, beta induced Tgfbitransforming −9.38 46 395 30 1815 1663 growth factor, beta inducedConsti tutive: Cd44 CD44 antigen 1.30 416 688 123 308 403 Cd44 CD44antigen −1.41 2061 5511 1508 2911 2937 Cd44 CD44 antigen −2.12 1826 56712410 4120 3991 Vcam1 vascular cell −1.94 193 326 1496 224 179 adhesionmolecule 1 Vcam1 vascular cell −2.16 132 347 778 152 162 adhesionmolecule 1 Vcam1 vascular cell −2.43 396 803 5015 545 388 adhesionmolecule 1 Vcam1 vascular cell −2.43 396 803 5015 545 388 adhesionmolecule 1 Total RNA was extracted from parental 3T3 cells and theircDNA-transfected subclones using TRIzol (Invitrogen-Molecular Probes),with relative transcript levels quantified in parallel by GeneChipanalysis (Affymetrix Mouse Genome 430 2.0 GeneChips; Affymetrix, SantaClara, CA). All quality parameters for the arrays were confirmed to bein the range recommended by the manufacturer. Linear relative expressionlevels are shown for each line. The results for reiterative probes oneach gene chip are shown.

Methods

Mice.

C57BL/6, IL-10^(−/−) (B6.129P2-Il10^(tmlCgn)/J) (Kuhn, et al.,Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75,263-274 (1993)), CD40^(−/−) (B6.129P2-CD40^(tmlKik)/J), and MD4(C57BL/6-Tg(TghelMD4)4Ccg/J) mice were from the Jackson Laboratory (BarHarbor, Me.). MHC-II^(−/−) (B6.129-H2-Ab1^(tmlJae)B2m^(tmGru)N17) mice(Taconic Farms, Inc., Hudson, N.Y.) were as described (Grusby, M. J. etal. Mice lacking major histocompatibility complex class I and class IImolecules. Proc. Natl. Acad. Sci. USA 90, 3913-3917 (1993)). CD19^(−/−)mice were backcrossed onto the C57BL/6 background for 14 generations.See Sato, S., Ono, N., Steeber, D. A., Pisetsky, D. S. & Tedder, T. F.CD19 regulates B lymphocyte signaling thresholds critical for thedevelopment of B-1 lineage cells and autoimmunity. J. Immunol. 157,4371-4378 (1996) and Sato, S., Steeber, D. A., Jansen, P. J. & Tedder,T. F. CD19 expression levels regulate B lymphocyte development: humanCD19 restores normal function in mice lacking endogenous CD19. J.Immunol. 158, 4662-4669 (1997). IL-21R^(−/−) mice were as described. SeeOzaki, K. et al. A critical role for IL-21 in regulating immunoglobulinproduction. Science 298, 1630-1634 (2002). TCR^(MOG) transgenic mice((Bettelli, E. et al. Myelin oligodendrocyte glycoprotein-specific Tcell receptor transgenic mice develop spontaneous autoimmune opticneuritis. J. Exp. Med. 197, 1073-1081 (2003)) Thy1.2⁺, provided by Dr.V. K. Kuchroo, Harvard Medical School, Boston, Mass.) were crossed toC57BL/6.Thy1.1 mice to generate Thy1.1-expressing T cells. All mice werebred in a specific pathogen-free barrier facility and used at 6-12 wksof age. The Duke University Animal Care and Use Committee approved allstudies.

Cell Preparation.

Single-cell suspensions from spleens and peripheral lymph nodes (pairedaxillary and inguinal) were generated by gentle dissection, with thecells passed through 70-mm cell strainers (BD Biosciences, San Diego,Calif.) followed by percoll gradient (70/37%) centrifugation.Lymphocytes were collected from the 37:70% interface and washed. MACS(Miltenyi Biotech, Auburn, Calif.) was used to purify lymphocytepopulations according to the manufacturer's instructions. CD19mAb-coated microbeads and CD4⁺ T cell isolation kits (Miltenyi Biotech)were used to purify B cells and CD4⁺ T cells, respectively. Whennecessary, the cells were enriched a second time using a fresh MACScolumn to obtain >95% cell purities.

Immunofluorescence Analysis.

FITC-, PE-, PE-Cy5-, PE-Cy7-, or APC-conjugated CD1d (1B1), CD4(H129.19), CD5 (53-7.3), CD19 (1D3), B220 (RA3-6B2), and Thy1.1 (OX-7)mAbs were from BD Biosciences. PE-conjugated IL-21R (4A9) mAb was fromBioLegend (San Diego, Calif.). Intracellular staining used mAbs reactivewith IL-10 (JES5-16E3), IL-17 (17B7), and IFN-γ (XMG1.2) (all fromeBioscience) and Cytofix/Cytoperm kits (BD Biosciences). Backgroundstaining was assessed using non-reactive, isotype-matched control mAbs(Caltag Laboratories, San Francisco, Calif.). For two- to six-colorimmunofluorescence analysis, single cell suspensions (10⁶ cells) werestained at 4° C. using predetermined optimal mAb concentrations for 20min as described. See Yanaba et al. 2008, Immunity 28, 639-650;Matsushita et al. 2008, J. Clin. Invest. 118, 3420-3430; Matsushita etal. 2010, J. Immunol. 185, 2240-2252; Matsushita and Tedder 2011,Methods Mol. Biol. 677, 99-111; and Zhou et al. 1994, Mol. Cell. Biol.14, 3884-3894. Blood erythrocytes were lysed after staining using FACS™Lysing Solution (Becton Dickinson, San Jose, Calif.).

B cell intracellular IL-10 expression was visualized byimmunofluorescence staining and analyzed by flow cytometry as described.See Yanaba et al. 2008, Immunity 28, 639-650 and Matsushita and Tedder2011, Methods Mol. Biol. 677, 99-111. Briefly, isolated leukocytes orpurified cells were resuspended (2×10⁶ cells/ml) in complete medium(RPMI 1640 media containing 10% FCS, 200 μg/ml penicillin, 200 U/mlstreptomycin, 4 mM L-Glutamine, and 5×10⁻⁵ M 2-mercaptoethanol, all fromGibco, Carlsbad, Calif.) with LPS (10 μg/ml, Escherichia coli serotype0111: B4, Sigma), PMA (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma)and monensin (2 μM; eBioscience) for 5 h in 48-well flat-bottom plates.In some experiments, the cells were incubated for 48 h with an agonisticanti-mouse CD40 mAb (1 μg/ml; HM40-3 mAb; BD Pharmingen) as described.Yanaba et al. 2009, J. Immunol. 182, 7459-7472. For IL-10 detection, Fcreceptors were blocked with mouse Fc receptor mAb (2.4G2; BDPharMingen), and dead cells were detected using a LIVE/DEAD® FixableViolet Dead Cell Stain Kit (Invitrogen-Molecular Probes) before cellsurface staining. Stained cells were fixed and permeabilized using aCytofix/Cytoperm kit (BD PharMingen) according to the manufacturer'sinstructions and stained with PE-conjugated mouse anti-IL-10 mAb.Splenocytes from IL-10^(−/−) mice served as negative controls todemonstrate specificity and to establish background IL-10 staininglevels. For T cell intracellular cytokine staining, lymphocytes werestimulated in vitro with PMA (50 ng/ml; Sigma, St. Louis, Mo.) andionomycin (1 μg/ml; Sigma) in the presence of Brefeldin A (BFA, 1 μl/ml;eBioscience) for 5 h before staining. Viable cells with the forward andside light scatter properties of lymphocytes were analyzed using aFACScan flow cytometer (Becton Dickinson) or BD FACSCanto™ II (BDBiosciences).

In Vitro B Cell Cultures.

Purified splenic B cells (1×10⁶/ml) were cultured in RPMI 1640 mediumcontaining 10% FBS, 2 mM L-Glutamine, penicillin (100 I.U./ml),streptomycin (100 μg/ml), and 50 μM 2-mercapthoethanol, and eitherrecombinant IFN-γ (10 ng/ml□□ IL-4 (2 ng/ml), IL-6 (10 ng/ml) or IL-21(100 ng/ml) (from e-Bioscience); TGF-β (10 ng/ml), IL-10 (10 ng/ml), orIL-12 (10 ng/ml) (from R&D systems, Minneapolis, Minn.); or IL-23 (20ng/ml) and IL-27 (100 ng/ml) (Biolegend), or LPS (10 μg/ml) before B10cell numbers and culture supernatant fluid IL-10 concentrations weredetermined. IL-10 concentrations were determined by ELISA. In separateexperiments, purified spleen B cells were cultured with NIH-3T3 cellsexpressing CD154 and BLyS as described with exogenous recombinant IL-4(2 ng/ml) or IL-21 (10 ng/ml) added to the cultures. Nojima et al. 2011,Nature Comm. 2, 465 and Tedder et al. in Leukocyte Typing V: White CellDifferentiation Antigens. Vol. 1 (eds S. F. Schlossman et al.) 483-504(Oxford University Press, 1995). For adoptive transfer experiments,cultured CD5⁺ and CD5⁻ B cells were purified by cell sorting(FACSVantage SE, Becton Dickinson), with purities of 95-98%. Afterpurification, 1×10⁶ cells were immediately transferred i.v. into eachrecipient mouse. In some experiments, CD40 mAb (clone HM40-3; hamster,no azide/endotoxin-free, BD Pharmingen, San Jose, Calif.) was added tocultures where indicated.

EAE Induction.

EAE was induced in 6- to 8-week-old female mice by subcutaneousimmunization with 100 μg of MOG₃₅₋₅₅ peptide (MEVGWYRSPFSRVVHLYRNGK;NeoMPS, San Diego, Calif.) emulsified in CFA containing 200 μg ofheat-killed Mycobacterium tuberculosis H37RA (Difco, Detroit, Mich.) onday 0. See Matsushita et al. 2008, J. Clin. Invest. 118, 3420-3430 andMatsushita et al. 2010, J. Immunol. 185, 2240-2252. Additionally, micereceived 200 ng of pertussis toxin (List Biological Laboratories,Campbell, Calif.) i.p. in 0.5 ml of PBS on days 0 and 2. Clinical signsof disease were assessed daily with a 0 to 6 point scoring system: 0,normal; 1, flaccid tail; 2, impaired righting reflex and/or gait; 3,partial hind limb paralysis; 4, total hind limb paralysis; 5, hind limbparalysis with partial forelimb paralysis; 6, moribund state, asdescribed. Fillatreau et al. 2002, Nat. Immunol. 3, 944-950. Moribundmice were given disease severity scores of 6 and euthanized.

Adoptive Transfer Experiments.

B cells from naïve mice or mice with EAE (day 28) were first enrichedusing CD19 mAb-coated microbeads, stained for cell surface CD19, CD1dand CD5 expression, with CD1d^(hi)CD5⁺ and CD1d^(lo)CD5⁻ B cellspurified by cell sorting as described with purities of 95-98%. SeeMatsushita 2011, Methods Mol. Biol. 677, 99-111 and Yanaba et al. 2008,Immunity 28, 639-650. After purification, the CD1d^(hi)CD5⁺ orCD1d^(lo)CD5⁻ B cells (1×10⁶) were immediately transferred i.v. intorecipient mice, with B10 cells representing 13-20% and <0.1% of thetransferred cells. In some experiments, donor Thy1.1 CD4⁺ T cells wereisolated from pooled spleens and lymph nodes of TCR^(MOG) transgenicmice, then labeled with CFSE Vybrant™ CFDA SE fluorescent dye (5 μM;CFSE; Invitrogen) and transferred i.v. (5×10⁶/mouse) into Thy1.2congenic recipients. Five days after adoptive transfer, the TCR^(MOG)CD4⁺ T cells were assessed by flow cytometry.

Statistical Analysis.

All data are shown as means (±SEM). The significance of differencesbetween sample means was determined using the Student's t test.

1.-21. (canceled)
 22. (canceled)
 23. (canceled)
 24. A method of treatinga subject having an autoimmune disorder comprising administering atherapeutically effective amount of a composition comprising B10 cellscultured in vitro with IL-21, wherein the B10 cells are more than 85% ofthe total B cells in the composition and wherein said B10 cells arecapable of producing IL-10 to a subject in need of treatment for anautoimmune disorder.
 25. The method of claim 24, wherein the autoimmunedisease is selected from multiple sclerosis, lupus, arthritis,inflammatory bowel disease and scleroderma.
 26. The method of claim 24,wherein the autoimmune disease is selected from allergic contactdermatitis, allergic reactions to drugs, alopecia areata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid and associatedskin diseases, cardiomyopathy, Celiac disease, Celiac sprue-dermatitis,chronic fatigue immune dysfunction syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn'sdisease, cutaneous necrotizing venulitis, discoid lupus, erythemamultiforme, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic/autoimmune thrombocytopenia purpura (ITP), immunologic lungdisease, immunologic renal disease, IgA neuropathy, juvenile arthritis,lichen planus, Meniere's disease, mixed connective tissue disease, type1 or immune-mediated diabetes mellitus, myasthenia gravis,pemphigus-related disorders (e.g., pemphigus vulgaris), perniciousanemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, rheumatic fever, sarcoidosis, Sjögren's syndrome, stiff-mansyndrome, spondyloarthropathies, systemic lupus erythematosis (SLE),lupus erythematosus, systemic vasculitis, takayasu arteritis, temporalarteristis/giant cell arteritis, thrombocytopenia, thyroiditis,ulcerative colitis, uveitis, vasculitides such as dermatitisherpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.
 27. Amethod of treating a subject to prevent or treat organ, tissue or celltransplant rejection or associated chronic graft versus host disease ortreating a subject receiving recombinant, therapeutic or xenogeneicprotein(s) comprising administering a therapeutically effective amountof a composition comprising B10 cells cultured in vitro with IL-21,wherein the B10 cells are more than 85% of the total B cells in thecomposition and wherein said B10 cells are capable of producing IL-10 toa subject in need of treatment for transplant rejection, graft versushost disease or other disorder associated with receipt of a transplantor protein treatment.
 28. A method of treating a subject having anallergic disorder or inflammatory disorder comprising administering atherapeutically effective amount of a composition comprising B10 cellscultured in vitro with IL-21, wherein the B10 cells are more than 85% ofthe total B cells in the composition and wherein said B10 cells arecapable of producing IL 10 to a subject in need of treatment forallergies or for inflammation.
 29. (canceled)
 30. The method of claim28, wherein the inflammatory disorder is selected from asthma,encephilitis, inflammatory bowel disease, chronic obstructive pulmonarydisease (COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacterial infections.
 31. (canceled)
 32. Themethod of claim 24, wherein the composition comprises autologous B10cells.
 33. (canceled)
 34. The method of claim 24, wherein thecomposition is administered after the onset of symptoms in the subject.35.-49. (canceled)
 50. The method of claim 24, wherein the compositioncomprises between 10⁶ and 10¹⁰ B10 cells.
 51. The method of claim 24,wherein the B10 cells were cultured in vitro with a CD40 agonist. 52.The method of claim 51, wherein the CD40 agonist is CD154, a fragment ofCD154, or antibody, aptamer or polypeptide, or fragment thereof reactivewith CD40.
 53. The method of claim 24, wherein the B10 cells werecultured in vitro with a B cells survival promoter.
 54. The method ofclaim 53, wherein the B cell survival promoter is selected from at leastone of feeder cells, BAFF (BLyS), BAFF fragments, APRIL, CD22 ligand,CD22 monoclonal antibody, or fragments thereof.
 55. The method of claim24, wherein the B10 cells were cultured on feeder cells expressing aCD40 agonist and a B cell survival promoter.
 56. The method of claim 55,wherein the feeder cells are fibroblast, endothelial cells, epithelialcells, keratinocytes, melanocytes, or other mesenchymal or stromalcells.
 57. The method of claim 24, wherein the B10 cells were culturedin vitro with IL-4.
 58. The method of claim 28, wherein the compositioncomprises autologous B10 cells.
 59. The method of claim 28, wherein theB10 cells were cultured in vitro with a CD40 agonist.
 60. The method ofclaim 28, wherein the B10 cells were cultured in vitro with a B cellssurvival promoter.
 61. The method of claim 28, wherein the B10 cellswere cultured in vitro with IL-4